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Seminars in Cancer Biology 21 (2011) 377–384

Contents lists available at SciVerse ScienceDirect

Seminars in Cancer Biology

journa l homepage: www.e lsev ier .com/ locate /semcancer

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he Myc/macrophage tango: Oncogene-induced senescence, Myc style

oyoung Leea,b, Clemens A. Schmitta,b,∗, Maurice Reimanna

Charité-Universitätsmedizin Berlin/Molekulares Krebsforschungszentrum der Charité – MKFZ, 13353 Berlin, GermanyMax-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany

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eywords:poptosisymphomaacrophagesouse Modelsycncogenesenescence

a b s t r a c t

Ras/Raf-prototypic oncogenes induce cellular senescence, a terminal cell-cycle arrest, as a default cellu-lar safeguard program, while oncogenic Myc is known to rather promote apoptosis as the prime failsafemechanism. We review and discuss here evidence for Myc-induced senescence – which is detectableto a limited degree as a cell-autonomous, direct response to Myc action, but occurs predominantly in anon-cell-autonomous fashion via crosstalk of the oncogene-driven cell population with non-neoplasticbystanders, namely cells of the host immune system, prompting them to release pro-senescent cytokinesthat strike back onto adjacent proliferating tumor cells. In particular, we discuss how Myc-evoked apo-

GF-� ptosis serves as a signal for macrophage attraction and activation, followed by the secretion of TGF-� asa cytokine that is capable of terminally arresting Myc-driven lymphoma cells without causing furtherDNA damage and without launching a senescence-associated, pro-inflammatory, and, therefore, poten-tially detrimental cytokine response in the target population. In essence, non-cell-autonomous but stilloncogene-orchestrated senescence is a functionally relevant, robustly tumor-suppressive principle withcritical implications for conceptually novel anti-cancer therapies in the clinic.

. Introduction

By stimulating aberrant proliferation and cell growth, proto-ypic oncogenes such as Myc and Ras evoke checkpoint-governedelf-defense mechanisms as a cellular countermeasure. Suchell-autonomous safeguard responses, namely senescence (“self-rresting”), apoptosis (“self-killing”), or autophagy (“self-eating”)rotect the organism against the uncontrolled expansion of poten-ially harmful cells. Simultaneously, these protective barriersmpose selective pressure to acquire mutations in tumor sup-ressor moieties, which, in turn, allow to overcome these failsafeechanisms, thereby collaborating with the initiating oncogene to

ventually drive cellular transformation [1]. What kind of cellu-ar failsafe mechanism a certain activated oncogene will actuallynitiate, depends on the orchestrated downstream signaling cas-ades but also on the cell type-specific properties. When expressedn fibroblasts, activated Ras/Raf oncogenes promote cellular senes-ence [2,3], while Myc is known as a pro-apoptotic oncogene [4,5].

pon their activation, Myc and Ras boost, at least temporarily,ell-cycle progression accompanied by excessive DNA replicationaltress, thus, leading to stalled replication forks as well as increased

∗ Corresponding author at: Charité-Universitätsmedizin Berlin/Molekulares Kreb-forschungszentrum der Charité – MKFZ, 13353 Berlin, Germany.el.: +49 30 450 553 896; fax: +49 30 450 553 986.

E-mail address: clemens.schmitt@charite.de (C.A. Schmitt).

044-579X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2011.10.002

© 2011 Elsevier Ltd. All rights reserved.

production of reactive oxygen species, both prompting a DNA dam-age response, which may subsequently trigger either one of the twoultimate failsafe effector programs senescence or apoptosis (Fig. 1).In this review, we will discuss how the initiating oncogene and itscellular downstream signaling, but also adjacent non-neoplasticcells collectively control the ultimate fate of the tumor cell pop-ulation through the promotion of cellular senescence.

2. Classic oncogene-induced senescence

Cellular senescence is a terminal growth arrest in the G1 phaseof the cell-cycle, featuring characteristic morphological, biochem-ical, and epigenetical changes [6,7]. Senescent cells are, in general,flatter and larger than normal proliferating cells, and their cyto-plasm is filled with vacuoles. Although arrested, senescent cellsare metabolically active and have a distinct gene expression pro-file. For example, a staining reaction that visualizes increased�-galactosidase activity at an acidic pH (senescence-associated-�-galactosidase, SA- �-gal) is used as a common marker for senescentcells [8] that also stain negative for the proliferation marker Ki67and no longer synthesize DNA (as one can experimentally demon-strate by lack of bromodesoxyuridine incorporation).

Reflecting the senescence-inducing condition, there are two

types of senescence, namely “replicative” and “premature” senes-cence. They share most of the specific senescence features, althoughthe initiating triggers are different. Replicative senescence wasfirst described about 50 years ago by Hayflick and Moorhead [9]

378 S. Lee et al. / Seminars in Cancer Biology 21 (2011) 377–384

Fig. 1. Prototypic oncogene-induced senescence (OIS) by Ras/Raf/Mek. Activated Ras/Raf/Mek oncogenes damage DNA, thereby triggering cellular DNA damage response(DDR) signaling involving ATM/ATR kinases and various components of the double strand break (DSB) repair machinery. Signals from unresolved DSB are relayed to thetumor suppressor p53, PML and pRB, which eventually promote a dynamic process of local, senescence-associated heterochromatin foci (SAHF) formation with the help ofhistone methyltransferases (such as Suv39h1) in the vicinity of E2F-responsive target genes, thereby transcriptionally silencing E2F-dependent S-phase genes. Moreover,p ve pro“ nesce( of My

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ersistent DSB may also trigger a second senescence-associated response, the massisenescence-associated secretory phenotype [SASP]”), considered to reinforce the seROS) and DNA replication stress like Ras/Raf-type oncogenes, and, a small fraction

s an ultimate growth arrest of human diploid cells after a cer-ain number of passages, later found to be caused by telomerehortening [10,11]. Premature senescence is named so becausef it is executed independently of telomere erosion, thereforebefore” the due time of a cell’s age or finite lifespan, and rep-esents and acutely, stress-inducible form of a terminal growthrrest. It was first reported as a response to viral infection [12],ut caught particular attention when it was found to be induced byctivated oncogenes such as Ras and Raf [2,3]. Furthermore, pre-ature senescence can also be induced by DNA damage, produced

y anti-cancer treatment modalities such as chemotherapy or ion-zing radiation [7]. Importantly, senescent cells are metabolicallyctive, suggesting an additional, active role in tumor developmentnd treatment responses. In this review, we will discuss prematureellular senescence (from now on, just ‘senescence’) in response toell-autonomous and non-cell-autonomous signals and the impli-ation of senescence in tumor development and therapy.

As described above, activation of the mitogenic Ras/Raf/Mek-ype oncogenes induces senescence in vitro. It is broadly acceptedhat mitogenic oncogenes such as Ras and Raf activate a DNAamage response (DDR)[13–15] by increasing the production of

ntracellular reactive oxygen species (ROS) [16], and by evokingNA damage as a consequence of initial hyperreplication that over-helms proper DNA synthesis and results in stalled replication

orks (Fig. 1). The DDR-mediating ATM and ATR kinases activate, viadditional downstream kinases, the tumor suppressor p53, whichperates as a central regulator of senescence and apoptosis. Onhe other hand, Ras/Raf – MAPK signaling leads to the inductionf p16INK4a, a CDK4 and CDK6 inhibitor, thereby, blocking hyper-hosphorylation of the Retinoblastoma protein (pRb), the majoruardian of the G1/S transition. Hypophosphorylated pRb, in com-lex with E2F transcription factors, is directed to E2F-responsivelements in promoters of S-phase-driving genes, thus bringingRb-bound histone modifiers such as HDAC (histone deacetylase)ctivities or the histone H3 lysine 9 methyltransferase Suv39h1 inheir vicinity. The specific signals that instruct this machinery toxecute local, senescence-associated heterochromatin foci (SAHF)ormation and silencing of E2F target genes [17] as compared to thescillating role of pRb in conjunction with chromatin-modifyingactors during regular cell-cycle progression or temporary arrest

onditions are not fully understood yet. Several Ras downstreamathways contribute to the terminal cell-cycle arrest: first of all,he mitogen-activated protein kinase (MAPK) cascade [18], andlso an attenuation of the initially hyperactivated PI3 kinase/Akt

duction of largely pro-inflammatory cytokines and other secretable factors (termednt arrest. Notably, the Myc oncogene is also known to evoke reactive oxygen speciesc-activated cells directly enter senescence in a cell-autonomous fashion.

pathway, promoting nuclear translocation and activation of FoxOtranscription factors [19] – whereby their individual relevancemight vary depending on the cellular context. Given the lack ofa senescence-defining marker or assay (in contrast to the multi-tude of markers established to detect apoptosis), a matrix of thejust mentioned senescence-related cell-cycle regulators includingp16INK4a, p21CIP1, p53, DDR mediators, and also chromatin remod-eling factors are collectively used as senescence indicators [20,21][22].

Following the observation of oncogene-induced senescence(OIS) in vitro, several groups simultaneously reported evidenceof OIS, and particularly of its tumor suppressive function, in vivo[23–25]. By examining BRAFV600E-positive nevus cell nevi, benignskin lesions that may stay arrested for years, but, in rare cases,progress at some point into a malignant melanoma, Daniel Peeperand colleagues found that the halted proliferation of nevi coincidedwith signs of cellular senescence, positive staining of SA-�-gal andp16INK4a, as well as H3K9 trimethylation-marked heterochroma-tinization [23]. In a mouse lung adenoma/adenocarcinoma modeldriven by endogenous Ras activation, Manuel Serrano’s groupdetected arrested cells with similar features of cellular senes-cence [25]. Using a transgenic mouse model where oncogenic Rasis activated in cells of the hematopoietic system, our own workunveiled that inactivation of senescence by genomic deletion ofSuv39h1 permitted B-cell lymphoma development, demonstrat-ing that senescence has tumor-suppressive potential in Ras-driventumorigenesis[24]. In addition, Pier Paolo Pandolfi’s group showedacute induction of cellular senescence in a setting where not pri-marily an activated oncogene but loss of a tumor suppressor – PTENin this particular model – drives full-blown progression to malig-nancy not before additional lesions such as inactivation of p53 areco-acquired to license bypass of senescence [26].

Of note, most of these findings and underlying mechanismsrelate to Ras/Raf-type oncogenes, for which the concept of OISwas inaugurated in the first place [2,3], while other prototypiconcogenes, such as c-Myc, are known to provoke apoptosis as theprimary cellular failsafe response [4,5]. Given the fact that the Myconcogene promotes ROS induction and DNA damage as well [27,28],we tested and found that Myc, especially under conditions in whichapoptosis is blocked, might trigger senescence as well (see below

[29]). Intriguingly, our study led to the observation and mechanisticexplanation how Myc drives a hitherto unknown type of prema-ture senescence that requires the cooperation of non-neoplasticbystander cells in the tumor stroma.

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. The senescence-associated secretory phenotype

Senescent cells are viable and can interact with other (non-enescent) tumor cells and non-malignant stroma cells, particularlyia the massive induction of secretable factors. Recent publica-ions focusing on this group of factors, collectively dubbed assenescence-associated secretory phenotype (SASP)” changed ouriew of senescence as a mere stress-inducible cell-autonomous fail-afe program to a principle with far-reaching environmental andven remotely impacting organismic implications.

In an effort to screen for genes required for OIS, Michael Green’sroup transduced human primary foreskin fibroblasts (PFF) thatere forced to senesce in response BRAF V600E expression, withuman shRNA library pools. From the non-senescent population,andidate shRNA sequences which allowed cells to bypass BRAF-nduced senescence, were identified. One of them, insulin growthactor binding protein 7 (IGFBP7) is a secreted protein, which isurrently subject of a controversial debate to whether or not it mayontribute to senescence induction in cells with BRAF activation inn autocrine/paracrine fashion [30,31]. In a similar approach, thehemokine receptor CXCR2 (also known as IL8RB) was found in anhRNA screening for genes that extend the lifespan of the humaniploid fibroblast (HDF) line IMR-90. While ectopic expressionf CXCR2 induced senescence, cells undergoing OIS were showno secrete CXCR2-binding chemokines, which, in turn, upregu-ate CXCR2 expression, thereby closing a circuit of senescenceelf-amplification [32]. Using combined genetics and bioinformaticnalyses, Daniel Peeper and colleagues reported opposing roles of aajor pro-inflammatory SASP cytokine, interleukin-6 (IL-6), which

hey identified as the critical SASP component that accounted fornhanced proliferation of susceptible hybridoma cells in a het-rologous setting, while reinforcing cellular senescence via anntracellular short-cut that involves induction of the cell-cyclenhibitor p15INK4B [33].

Interestingly, work by Judith Campisi’s group primarily con-ucted in fibroblasts characterized SASP (mainly IL-6 and IL-8)ather as a response to genotoxic stress, engaging DNA dam-ge response proteins such as ATM, NBS1, CHK2, than as aenescence-dependent condition [34,35]. They even observed aarticularly strong induction of SASP in cells lacking p53, thus

lacing p53 as a mediator of senescence but suppressor ofASP. Beyond the – certainly context-dependent – role of p53n potentially restraining a SASP response, additional findings ofampisi and colleagues elegantly underscore how SASP act as a

ig. 2. Myc’s anti-senescence function. Myc, by and large, causes little strictlyell-autonomously governed senescence, because additional Myc-controlled down-tream pathways rather antagonize senescence. Myc induces genes conferringell-cycle progression (e.g. CycD1 and CDK4), and represses cell-cycle inhibitors (e.g.15INK4b, p21CIP1). Moreover, Myc drives the expression of target genes (e.g. WRN,ERT), whose inactivation unveils senescence under the condition of active Myc. Inddition, Myc possesses immunosuppressive potential by impairing antigen pro-essing and presentation, thereby preventing T-cells from recognizing tumor cells,nd, thus, from releasing pro-senescent secretable factors in closest proximity tohe Myc-driven cells.

iology 21 (2011) 377–384 379

non-cell-autonomous messaging system that instructs profoundremodeling of the tumor and its environment, for instance relatedto inflammation, epithelial–mesenchymal transition and invasive-ness.

A critical association of SASP production and host immune-mediated clearance of senescent tumor cells was reported by Xueet al. from Scott Lowe’s laboratory in a Ras-driven model of livercancer that formed in the absence of functional p53 due to aconditional p53-targeting siRNA [36]. In this system, acute re-activation of endogenous p53 expectedly induced senescence ofthe manifest tumor cells. Although the cells did not primarilydie, the authors observed subsequent regression of the macro-scopic tumor lesions – which was surprising since senescenceshould have resulted in a stabilized tumor burden. Intriguingly,the senescence-accompanying inflammatory cytokine productionapparently triggered an innate immune response that contributedto the clearance of senescent tumor cells in vivo.

4. Myc-related types of cellular senescence

4.1. Myc effector functions rather protect cells from senescence

It is generally accepted that senescence is not the primeresponse to Myc activation [37]. From early on, it has beenknown that ectopic overexpression of Myc induces apoptosis, mostlikely by promoting excessive cell growth, i.e. aberrant cell-cycleprogression, ROS induction, metabolic dysregulation and otheraberrant proliferation-related features that all prompt checkpoint-mediated induction of cell death [38]. Myc-controlled downstreameffectors, which account for approximately 15% of the entire tran-scriptome, equip this oncogene with the capacity to “keep cellsin cycle” (Fig. 2) [39,40]. Myc induces genes involved in cell-cycle progression (e.g. Cyclin D1 and 2, CDK4) and increased cellsize or “biomass” (e.g. lactate dehydrogenase, ribosomal proteins,eIF4E, eIF2A). In addition, Myc represses cell-cycle inhibitors, forexample p15INK4B or p21CIP1, by binding to the transcription fac-tor Miz-1, thereby blocking Miz-1-mediated transactivation atthese respective promoters [41,42]. Moreover, elegant work byJoan Massague and colleagues suggested that Myc binding toMiz-1 prevents p53 and other activators from inducing p21CIP1,thereby switching the cell’s “failsafe mode” from cytostatic toapoptotic [43].

However, there are conditions in which Myc directly evokessenescence that become particularly evident when certainanti-senescent Myc effectors are selectively disabled (Fig. 2). Over-expression of Myc in conjunction with an inactivating mutation ofthe WRN gene (Werner syndrome gene, RecQ helicase), which playsan important role in the maintenance of telomeres, induced senes-cence that could not be suppressed by hTERT expression. SinceMyc activates WRN transcription, the authors concluded that WRNsuppresses Myc-induced senescence [44].

David Feldser and Carol Greider examined the role of an addi-tional telomerase defect (due to lack of the RNA component oftelomerase) in the E�-myc transgenic mouse B-cell lymphomamodel. Even when apoptosis was blocked by Bcl2 overexpression,late-generation telomerase-deficient E�-myc mice appeared to bewell protected against lymphoma formation, and this observa-tion could be explained by the induction of cellular senescence inresponse to critically short telomeres [45]. Notably, senescence inthis experimental setting is not directly linked to Myc action andrather occurred despite of Myc, although Myc-related induction of

ARF, and, thus, of p53, may have contributed to the phenotype [46].Physiologically, Myc seems to counter telomere-related replicativesenescence, since Myc directly transactivates TERT, the catalyticsubunit of telomerase [47].

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Work by Bruno Amati’s and Lars-Gunnar Larsson’s laboratoriesdentified CDK2 as another constraint of Myc-related senescence,howing that loss of this cyclin-dependent kinase sensitized cellsith constitutive Myc activation to a terminal cell-cycle arrest,hich was accompanied by induction of p21CIP1 and p16INK4a.ccordingly, CDK2-deficient E�-myc transgenic mice developed

ymphomas slower than CDK2-proficient mice, again uncover-ng the tumor-suppressive role of senescence in Myc-inducedumorigenesis [48]. Indicating a potential therapeutic value, phar-

acological inhibition of CDK2 function enhanced Myc-relatedellular senescence in various settings including cells lacking intact53 alleles. Thus, CDK2-dependent suppression of Myc-inducedenescence creates a special variation of “synthetic lethality” whereyc overexpression as the first condition renders specifically

ancer cells susceptible to growth-terminating pharmacologicalnterference with CDK2 as the secondary hit, while CDK2 inhibitions not essential in cells without constitutive Myc activation.

.2. Myc may induce senescence in a cell-autonomous fashion

Our own experiments demonstrated that overexpression of c-yc in primary mouse B-cells results in quantitative cell death,

ut, in addition, also a smaller fraction of senescent cells that wasargely reduced in B-cells lacking suv39h1 or p53 alleles [29]. Moremportantly, we were able to show that E�-myc transgenic lym-homas which formed in the absence of functional p53 and werengineered to overexpress Bcl2 to block apoptosis independentlyf p53 were still susceptible to the tumor-suppressive potentialf acutely reactivated p53 [29]. Conditional re-activation of p53 –tilizing tamoxifen-inducible p53-estrogen receptor fusion alleles49] – induced senescence in manifest Bcl2-protected myc trans-enic lymphoma cells, but not in non-transgenic B-cells in vitro,hereby providing first genetic evidence that global Myc action

ay indeed drive senescence in a setting where no specific anti-enescent Myc effector function had been selectively disabledeforehand. Similarly, acute induction of functional p53 in miceearing bcl2-infected lymphomas produced massive lymphomaell senescence in situ. The observation that Myc-induced senes-ence could be ablated by exposure to a ROS scavenger and/oro an Atm/Atr inhibitor further suggested that Myc evokes cellu-ar senescence in a cell-autonomous setting predominantly via aOS-initiated DDR (Fig. 1).

For a balanced view on Myc’s potential to directly launch a cel-ular senescence response, it should not be overlooked that it is arime capability of Myc to directly transform mouse cells in coop-ration with oncogenic Ras, thus canceling Ras-induced senescences an essential barrier against transformation [50]. Likewise, Mycas been shown to override cellular senescence induced by the ini-iation of translation factor eIF-4E [51]. In essence, direct inductionf cellular senescence by Myc is probably restricted to a minorityf cells that – for various reasons – did not primarily respond withpoptosis. Settings, in which apoptosis is a priori not an option (seebove), are, however, much more susceptible to the pro-senescentotential of Myc.

.3. Myc-induced senescence via recruitment ofon-oncogene-driven bystander cells

Despite a growing body of evidence for a – certainly limitedrole of Myc in the cell-autonomous induction of senescence as

iscussed above, there is clear evidence that Myc-evoked cellu-ar failsafe responses favor apoptosis over arrest [43]. Accordingly,

poptosis has been identified as the critical p53-governed tumoruppressor function, which is, in turn, why p53 is selected againsturing Myc-driven lymphoma development in the E�-myc trans-enic mouse lymphoma model [52]. To clarify the contribution of

iology 21 (2011) 377–384

Myc-induced senescence in tumor suppression, we utilized the E�-myc transgenic mouse model and generated Suv39h1-proficientand deficient E�-myc offspring. As previously mentioned, thehistone methyltransferase Suv39h1 plays an essential role in Ras-driven settings of OIS, at least in mouse cells of hematopoietic origin[24]. Contradicting our assumption that senescence may play aminor role in Myc-induced tumorigenesis, mice lacking one or twosuv39h1 alleles presented with a dramatically accelerated tumoronset, when compared to a cohort of suv39h1+/+ mice. Notably,oncogene-induced apoptosis – measured as the high extent ofTUNEL-positive cells present in Myc-driven lymphomas at diag-nosis – remained unaffected by lack of suv39h1 alleles. However,we detected a significant proportion of senescent tumor cells inmanifest B-cell lymphomas of regular Suv39h1 status (hereafterreferred to as “control” lymphomas) based on double-stainingsfor SA-�-gal reactivity with either the proliferation marker Ki67or bromodesoxyuridine incorporation as an indicator for DNAsynthesis. In sharp contrast, Myc-related senescence was virtu-ally absent in Suv39h1-deficient lymphomas, indicating that OISutilizes a conserved, Suv39h1-controlled downstream machin-ery and represents a critical barrier not only during Ras [24]-,but also Myc-driven lymphomagenesis [29]. Further analysis ofgenome-wide microarray-based gene expression profiles obtainedin freshly isolated lymph node material from Suv39h1-proficientvs. Suv39h1-deficient lymphoma-bearing mice pointed towards arole for enhanced tumor growth factor-� (TGF-�) signaling in con-trol lymphomas as a potential senescence-promoting mechanism.Indeed, control, but not Suv39h1-deficient Bcl2-overexpressinglymphoma cells responded with SA-�-gal- and H3K9me3-positivecellular senescence and a firm growth arrest to TGF-� exposurein vitro. To confirm the pro-senescent role of TGF-� in vivo, weblocked its action in the E�-myc model system by utilizing theextracellular domain of the TGF-� type II receptor (T�R-II-ED) thatbinds to and neutralizes TGF-� in the vicinity of the receptor-releasing cell. When T�R-II-ED was stably expressed in E�-myctransgenic hematopoietic stem cell prior to their transplantationinto lethally irradiated recipients, lymphoma onset was dramati-cally accelerated and manifest lymphomas displayed diminishedsenescence. We identified tumor-associated macrophages (TAM)of the CD206-positive “M2” type [53] as the major source of cyto-static TGF-�, and suspected its release to occur upon phagocytosisof apoptotic lymphoma cells [54,55]. Accordingly, depletion of TAMin lymphoma-bearing mice by systemically providing liposomalclodronate as a macrophage poison, or by blocking Myc-inducedapoptosis via Bcl2 overexpression – which virtually abrogatedmacrophage infiltration at the lymphoma site – led to a signifi-cant decrease of tumor cell senescence. Vice versa, the adoptivetransfer of activated macrophages into lymphoma-bearing miceproduced a drastic increase of lymphoma cell senescence that wasaccompanied by an accumulation of the exogenously providedmacrophages at the tumor sites. Importantly, the recapitulationof this process in a single in vitro-experiment underscored theessential components: macrophages in a lymphoma/macrophageco-culture system became activated upon lymphoma cell apopto-sis caused by acute p53 re-activation, and promoted senescence ofapoptotis-blocked lymphoma cells only in the absence of a phar-macological TGF-� inhibitor (Fig. 3). Providing “proof-of-relevance”for these mouse model-derived results in the human condition,we uncovered a strong correlation between the proliferation sta-tus of diffuse large B-cell lymphomas (DLBCL, the largest andclinically most relevant entity in the group of aggressive B-celllymphoma, which, in addition, frequently presents with Myc acti-

vation) samples and surrogate markers collectively recapitulatingthe mode of non-cell-autonomous senescence induction in themouse: although all DLBCL are characterized by high proliferativeactivity, we found the cohort of DLBCL samples defined by less

S. Lee et al. / Seminars in Cancer Biology 21 (2011) 377–384 381

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ig. 3. Non-cell autonomous Myc-induced senescence. Activation of Myc provokesre engulfed by host macrophages, which, in turn, get activated and secrete, amoenescence in Myc-driven lymphoma cells.

xtensive Ki67 staining to be characterized by more apoptosis, aigher extent of macrophage infiltration, activated TGF-� signal-

ng, and, ultimately, a much higher frequency of H3K9me3-positiveells indicative of cellular senescence. In essence, these observa-ions were well in accordance with the findings obtained in theenetically engineered settings of the E�-myc mouse lymphomaodel, suggesting that both senescence and apoptosis are indeed

inked via a macrophage-involving process, and, therefore, are bothrchestrated by the Myc oncogene in the tumor cell compartment.he close interconnection of oncogene-evoked apoptosis, TGF-�-eleasing action of infiltrating macrophages upon the ingestion ofpoptotic remainders, and the susceptibility of Myc-driven prolif-rating lymphoma cells to TGF-�-induced senescence underpins aomplex tumor-suppressive sequence whose components wouldave not been identified and functionally dissected without theelp of a tractable transgenic mouse lymphoma model (Fig. 3).

. Cell-autonomous and non-cell-autonomous senescencenduction following reduced Myc function

In a series of experiments using HDF engineered to harbornly one copy of c-myc, Guney et al. from John Sedivy’s labora-ory observed that overexpression of hTERT, otherwise leading tommortalization of normal HDF, produced senescence when com-ined with reduced c-Myc expression levels [56]. The senescencehenotype was accompanied by increased expression of p16INK4a

nd reduced expression of the polycomb member Bmi-1, whichcts as an inhibitor at the INK4a/ARF locus and is a transcriptionalarget of Myc. Therefore, global reduction of c-Myc expression lev-ls may – similarly to the acute induction of Myc expression [29]promote cellular senescence, albeit for different reasons, therebyointing towards Myc’s “double-edged” functional properties of aegative senescence regulator on one hand and of a pro-senescentOS-related stress inducer on the other hand.

Along the same lines, collaborative investigations by Martinilers and Dean Felsher discovered TGF-�-induced senescenceollowing Myc inactivation – mirroring our findings of non-cell-utonomous TGF-�-induced senescence as an indirect response toncogenic Myc action [29] – in the context of a specific conditionalouse model. Using a Myc mutant (V349D) that cannot bind toiz-1, and, thereby, cannot suppress cyclin-dependent kinase

nhibitors, the Eilers and Felsher groups showed that Myc antago-izes senescence in T-cell lymphomas, again supporting the view

f Myc as a oncogenic principle whose pro-senescent function isntrinsically countered by additional Myc-related properties. Inter-stingly, these Myc-V349D-driven lymphomas expressed highevels of TGF-�2 and TGF-�3, and senesced upon Myc inactivation

lar counteractions, predominantly apoptotic cell death. Apoptotic lymphoma cellsher factors, TGF-�. Critically increased local TGF-� levels are sufficient to induce

[57–59]. Their result suggested that Myc-related senescence –reflected by high expression levels of cell-cycle inhibitors such asp15INK4B and p21CIP1 and induced by TGF-� in the V349-mutantscenario – is normally actively suppressed by Myc. Lymphomaswith mutant Myc arose later compared to wild-type Myc-drivenlymphomas, because their expansion is slowed by a higher pro-portion of senescent cells. While both our E�-myc transgenicB-cell lymphoma model and this conditionally Myc-driven T-celllymphoma model converge at the finding that TGF-�-inducedsenescence is an important tumor-suppressive mechanism inMyc-related lymphomagenesis, it is noteworthy to stress thatboth systems present with little, directly Myc-attributable senes-cence, but exhibit a strong senescent phenotype either as theconsequence of Myc-inactivation in T-lymphoma cells, or, as anon-cell-autonomous feedback action to Myc-induced apoptosisin B-lymphoma cells that did not express TGF-� on their own.

Senescence onset in lymphoid tumor cells upon inactivationof Myc may also involve non-cell-autonomous components. Arecent study, again conducted by Dean Felsher and colleagues,identified CD4+ T-cells as critical mediator of cellular senescencefollowing oncogene inactivation in a Myc-driven mouse modelof T-cell acute lymphoblastic lymphoma [60]. When transplantedinto either CD4-deficient or RAG2−/− mice (lacking both matureB- and T-cells), these tumors failed to senesce after Myc wasturned off. Moreover, CD4+ T-cell action was associated with ablock of tumor angiogenesis, prompting the authors to investi-gate thrombospondin-1, a T-cell-expressed, secretable factor withprofound anti-angiogenic properties. Interestingly, T-cells lackingthrombospondin-1 and -2 were not capable of mediating lym-phoma cell senescence. Hence, both Myc-driven and Myc-deprivedlymphoid tumors enter senescence via a significant contributionof non-cell-autonomous components of the host immune sys-tem. While apoptotic E�-myc transgenic B-cell lymphomas attractinnate immune cells, namely macrophages, which subsequentlyengulf the apoptotic bodies [54,55,61] and promote lymphomacell senescence by release of TGF-�, it remains to be eluci-dated in greater detail why lymphoblastic T-cell lymphoma cellscrosstalk to CD4+ T-cells in response to Myc inactivation, to even-tually enter senescence conferred by T-cell-released cytokines,among them thrombospondin-1. Since Myc has been shown tolimit T-cell-governed immune recognition by suppressing NF-�Band interferon-related antigen presentation, it is well conceiv-able that restoration of immunogenicity upon Myc inactivation

provides the basis for pro-senescent T-cell action within thetumor cell compartment [62]. In essence, reduced Myc functionaccounts for senescence-promoting cell-autonomous and non-cell-autonomous biological consequences. Whether absolute Myc

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xpression below wild-type levels or rather their instant rela-ive decline establish the critical signal that eventually triggersro-senescent effector mechanisms is a question of important ther-peutic ramifications and will require future investigations.

. TGF-�-mediated induction of cellular senescence

TGF-� is known to induce cellular senescence in non-lymphoidells [63], but its role in cancer – acting as a tumor suppressorn early (pre-)neoplastic lesions, or promoting tumor growth and

etastasis in more advanced stages, and even enhancing malig-ant stemness – can be diverse [64–66]. How precisely TGF-�

nduces Suv39h1-dependent senescence in Myc-overexpressingells remains to be elucidated. Since we could not detect any inter-ction between Smad proteins, the downstream transducers ofGF-� receptor signaling, and Suv39h1, and did not find p15INK4B

r p21CIP1 to be clearly involved in TGF-�-induced senescence,e hypothesize Smad-independent collateral pathways to connect

GF-� action to the senescence-relevant control of the pRb/E2F-overned G1/S transition [64].

Importantly, TGF-�-induced senescence of Myc-driven lym-homa cells, although equally dependent on the Suv39h1-overned heterochromatinization effector machinery as previouslyeported for Ras-evoked senescence [24,29], differs from “clas-ic” OIS and senescence induced by DNA-damaging anticancergents or �-irradiation by the lack of additional DNA damage, i.e.n increased number of �-H2AX foci, following exposure to TGF-. Thus, unlike DDR signaling, which undoubtedly represents theommon upstream cascade linking oncogenic action to senescence13–15], TGF-� apparently produces tumor-restraining senescenceithout causing possibly mutagenic DNA lesions, thus rendering

imilar cytokines prototypic pro-senescent drugs with consider-ble clinical potential. This view is further supported by the facthat TGF-�-induced senescence is not accompanied by SASP, which,s a cocktail of predominantly pro-inflammatory cytokines, mightccount for a rather tumor-promoting chronic inflammatory con-ition at the tumor site [67]. Hence, TGF-�-induced senescence notnly represents an important tumor-controlling component in theontext of oncogenic Myc, but might very well serve as the naturalaradigm of future non-genotoxic pro-senescent therapies in thelinic.

Notably, the role of tumor-associated macrophages – the non-ell-autonomous source of TGF-� in the E�-myc lymphoma modelnd presumably in DLBCL as well [29] – is an issue of intense debate.n the field of malignant lymphomas, a high content of macrophagenfiltration is reportedly associated with rather poor prognosis inollicular lymphoma and Hodgkin’s lymphoma [68,69], but not inLBCL. Importantly, a stromal gene expression signature reminis-ent of macrophages and TGF-� action in particular was found in anndependent transcriptome-based investigation to predict superiorutcome in this entity [70].

. Discussion

.1. Oncogene-induced senescence, Myc style

Myc-instructed senescence shares many phenotypic and mech-nistic aspects of “classic” Ras/Raf-type OIS (e.g. involvement of theDR, p53, Rb, as well as histone modification and heterochromatin

ormation). Myc, like oncogenic Ras or Raf, drives, via increasedOS production and activated DDR signaling, cellular processes

hat are known to trigger cellular senescence [13–15,28]. How-ver, these mediators rather produce apoptosis in a Myc-governedetting, where directly Myc-attributable senescence – in contrasto apoptosis as the prime Myc-evoked failsafe program – has

iology 21 (2011) 377–384

little quantitative importance, because of numerous Myc targetfunctions that actively suppress senescence in many cell type- orcontext-dependent scenarios. While ROS or DDR signaling repre-sent upstream triggers also involved in Ras-induced senescence,senescence-relevant downstream signaling networks substantiallydiffer between Ras and Myc – irrespective of the fact that the ulti-mate effector machinery, based on firm silencing of S-phase genesas a consequence of selective heterochromatinization, is shared byboth Myc- and Ras-related senescence. While Ras-induced senes-cence utilizes the MAP kinase pathway and depends on an initialhyperactivation of PI3 kinase/Akt signaling, which promotes pro-senescent FoxO activation upon its feedback attenuation [19], Mycsignaling is predominantly characterized by the intrinsic controlof “anti-senescence” effectors such as CDK4 or telomerase induc-tion or Miz-1 binding-related repression of p15INK4B or p21CIP1

expression. Moreover, the relative genetic bias towards the expres-sion of pro-apoptotic genes provides another explanation whyprimary senescence is rarely seen in Myc-hyperactivated scenar-ios: most of the cells would have died apoptotically before themore delayed failsafe alternative would become phenotypicallyand quantitatively visible [43]. In turn, directly pro-senescent Mycaction becomes particularly visible when Myc-evoked upstreamROS/DDR signaling meets a firm apoptotic block, thereby establish-ing a lasting stress signal that may shift the cellular responsivenesstowards senescence despite constitutive Myc expression levels.

The unique feature of “senescence, Myc style” is its dependencyon non-neoplastic bystanders, especially cellular components ofthe host immune system, as reported first of all for macrophagesin a “Myc on” setting, and, with a slightly different scope, for T-helper cells in a “Myc off” setting [29,60]. It is the “tango”, theintimate engagement, of two cell populations – tumor cells and hostimmune cells – to eventually execute an indirect type of tumor cellsenescence. Compared to primary, cell-autonomous senescence inresponse to Myc, these immune effector-mediated forms of senes-cence are of much greater functional relevance, as demonstratedfor the tumor-suppressive role of macrophages in E�-myc trans-genic B-cell lymphomagenesis or for CD4+ T-cell-mediated T-cellacute lymphoblastic lymphoma regression. While the intercellularsignals between “Myc off” lymphoma cells and T-cells remain to beelucidated, macrophage activation by “Myc on” lymphoma cells hasbeen shown to depend on apoptosis and engulfment of dead tumorcells as indispensable intermediate steps. Therefore, immune cell-mediated, especially, macrophage-driven senescence appears to bea process of critical “local proximity” – dependent on cell–cell inter-actions and effective only on short distances because of rapidlyvanishing cytokine gradients. Moreover, unlike DDR-relayed classicOIS, where an activated oncogene permanently supports high ROSlevels or constitutive DDR signaling, it is currently unclear whetherthe maintenance of TGF-� or thrombospondin-1-mediated senes-cence depends on the chronic provision of these secreted factors.Given the lack of a detectable increase of DNA damage foci andno SASP induction upon TGF-�, both of which may contribute toa sustained senescent phenotype, cytokine-triggered senescenceeither reflects a permanent condition because of secondary chro-matin remodeling processes, or is much less stable compared toclassic OIS. However, a less robust type of senescence does notnecessarily translate into a functionally less relevant form of thisfailsafe response: as reported for classic OIS [36], senescent cells getcleared by a variety of innate host immune cells, and, thus, are elim-inated from the pool of cells potentially giving rise to a relapse atsome later point. Although formally to be proven, TGF-�-senescentB-lymphoma cells are likely to face the same fate, hence, underscor-

ing the tumor-suppressive implications of this type of senescence,no matter if maintained for long periods of time or not. It will beimportant to test in this regard whether the lack of SASP, and, there-fore, senescence-associated chemokines capable of attracting host

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mmune cells, critically affects the ultimate fate of TGF-�-senescentumor cells.

The above mentioned cascade of “oncogene action-apoptosisxecution-host immune cell interference-cytokine secretion-enescence induction” provides a conceptually novel way ofumor suppression, where apoptosis and senescence can no longere viewed as separate mechanisms but are thoroughly inter-onnected. Interestingly, selective inactivation of Myc-provokedpoptosis anticipates reduced senescence, too. Since apoptoticefects are frequently acquired during Myc-driven transforma-ion, non-cell-autonomous induction of senescence will be lessronounced in those cases. However, reduced senescence as a con-equence of strictly apoptotic defects will alleviate the pressure toubsequently select for mutations in the senescence machinery asell, thereby preserving an intact senescence program that may be

ecruited by anti-cancer therapy, and, thus, might ultimately trans-ate into superior long-term outcome despite the compromisedpoptotic response to chemotherapy [71].

.2. Concluding remarks

It is still unclear whether, as a net value, senescence is aore desirable or rather harmful component of tumor biology

nd treatment responsiveness. On first sight, senescence and apo-tosis are two qualitatively comparable programs when vieweds safeguard mechanisms that enforce an ultimate cell-cycle exit,hereby preventing (pre-)malignant cells from further contribut-ng to the expansion of an eventually deleterious tumor mass.

hile genetic work conducted in mouse models or correlativetudies in patients either exposed to neo-adjuvant chemother-py prior to the removal of the remaining tumor mass or in theetastasized setting suggested a growth-controlling and long-

erm beneficial role of senescence [71–73], other features ofenescent cells, in particular their massively boosted secretionf largely pro-inflammatory cytokines raise concerns about sec-ndary implications of senescence that may promote, in someases, even more aggressive tumor relapses. Although TGF-� is notcytokine suitable to treat patients in the clinic for various rea-

ons, the dissection of a natural, Myc-initiated process to promoteenescence via macrophage-released TGF-� that occurs withoutdditional DNA damage provides a very appealing “guideline” ofow to tackle tumors with pro-senescent but non-genotoxic andon-SASP-inducing agents more effectively in the future.

onflict of interest

The authors declare that they have no conflict of interests.

cknowledgements

The authors thank members of the Schmitt lab for criticaliscussions. This work was supported by Deutsche Forschungsge-einschaft (TRR54), the Deutsche Krebshilfe, and the Experimental

nd Clinical Research Center of the Charité - Universitätsmedizinerlin and the Max-Delbrück-Center for Molecular Medicine.

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