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Control of senescence by CXCR2 and its ligands Juan C. Acosta, Ana O’Loghlen, Ana Banito, Selina Raguz and Jesús Gil* Cell Proliferation Group; MRC Clinical Sciences Centre; Faculty of Medicine; Imperial College; London United Kingdom
Abbreviations: CXCR2, chemokine receptor 2; PAI-1, plasminogen activator inhibitor 1; IGFBP-7, insulin-like growth factor binding protein 7; IL-6, interleukin 6; IL-8, interleukin 8; NFκB, nuclear factor-kappa B; C/EBPb, CCAAT enhancer binding protein beta; SAHF, senescence-associated heterochromatin foci; SASP, senescence-associated secretory phenotype; HCC, hepatocellular carcinoma; VEGF, vascular endothelial growth factor; OIS, oncogene-induced senescence; ROS, reactive oxygen species
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in contrast with quiescence, adding growth factors does not force re-entry of senescent cells into the cell cycle. Different possibilities have been proposed to explain the irreversibility of this arrest, namely chromatin modifications5 or the trigger of a cytokinetic block as a second barrier in cellular senescence.6 Recent evidence suggests that maybe some of the factors secreted by senescent cells could also contribute to establish an additional layer of irreversibility by reinforcing the growth arrest.7,8 As mentioned above, senescent cells present a complex ‘senescence-associated secretory phenotype’ or SASP (a term coined by Judy Campisi). Among the multiple factors secreted by senescent cells, there are extracellular proteases, matrix components, growth factors, pro-inflammatory cytokines and chemokines.2 Through secretion of all these factors, senescent cells can alter the tissue microenvironment and influence neighboring cells in different manners. For example, studies by Campisi’s group have shown that senescent fibroblasts exert pro-malignant effects over transformed epithelial cells. They can promote the growth of epithelial cancer cells, enhance their tumorigenic properties9 and alter their differentiation.10 In addition senescent cells can also promote angiogenesis, an effect mediated by their increased production of VEGF.11 More recently, in addition to these heterotypic tumor-promoting effects, the SASP has been proven necessary to restrain tumor progression. In an elegant study, Xue et al., demonstrated that the regression of hepatocellular carcinomas (HCCs) upon reactivation of p53, is a consequence of a senescent response.12 Specifically, they showed that cytokines secreted by senescent cells in the incipient tumors triggered an innate immune response that contributes to clear the lesions and eventually limits tumor progression. A number of recent studies have shown that factors that are secreted by senescent cells contribute to reinforce their growth arrest (reviewed in ref. 13). Amongst them are the plasminogen activator inhibitor-1 (PAI-1), the insulin-like growth factor binding protein 7 (IGFBP7), IL-6 or CXCR2 ligands such as IL-8 or GROa.7,8,14,15 All of them cooperate to reinforce senescence although they do so by distinct mechanisms. PAI-1, which transcription is upregulated during senescence in a p53-dependent manner has been proposed to modulate senescence by controlling PI3K activity and cyclin D1 localization.14 IGFBP7 affects MAPK signaling,15 disrupting the negative feedback responsible of
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Senescence is an irreversible growth arrest with important physiological implications as it contributes to tumour suppression and may have a role in aging. During senescence, cells suffer profound phenotypic changes affecting amongst others cell morphology and chromatin structure. Senescent cells also undergo significant transcriptional changes, such as the increased production of a plethora of different secreted factors, which are the basis of the so-called senescence-associated secretory phenotype. While some of these factors have been previously shown to possess different pro-tumorigenic activities, we recently demonstrated that the secretion of CXCR2-binding chemokines (such as IL-8 or GROa) by senescent cells contribute to reinforce senescence via activation of the p53 pathway. Importantly, our data adds to that presented by several groups suggesting that also other factors secreted during senescence (such as PAI-1, IGFBP-7 or IL-6) contribute to the senescent response. Here, we discuss our findings in the context of the emerging role for secreted factors in regulating senescence through paracrine and/or autocrine mechanisms.
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Key words: CXCR2, senescence, IL-8, DNA damage, tumor suppressor, chemokines, secreted factors
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Senescence was first described as a highly irreversible growth arrest concomitant with the loss of proliferative potential in primary cells.1 Later studies have clearly established that a growing number of insults, the most prominent amongst them being aberrant oncogenic signaling, can also trigger a response that essentially phenocopies that of replicative senescence.2 Senescent cells have a characteristic flat and enlarged morphology, reorganize their chromatin in specific domains and despite being growth arrested remain metabolically active. In addition, senescent cells present profound changes in their transcriptional profiles.3,4 Interestingly, amongst the genes whose expression is upregulated during senescence there are a myriad of secreted proteins. The defining characteristic of senescence is that *Correspondence to: Jesús Gil; Cell Proliferation Group; MRC Clinical Sciences Centre; Faculty of Medicine; Imperial College; Hammersmith Campus; London W12 0NN United Kingdom; Tel.: +44.0.20.8383.8263; Fax: +44.0.20.8383.8306; Email:
[email protected] Submitted: 08/07/08; Accepted: 08/13/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6780 2956
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Role of CXCR2 on senescence
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maintaining oncogene-induced senescence (OIS).13 On the other hand, IL-6 controls C/EBPb activity8 which in turn regulates directly p15INK4b expression.16 Upregulation of p15INK4b results in increased SAHF formation and contributes to the growth arrest.8 In addition to this, our group identified in a genetic screen, that knocking down CXCR2 extends the lifespan of IMR-90 cells.7 In fact, depletion of CXCR2 expression also extended the lifespan of other human diploid fibroblasts (WI-38) and human mammary epithelial cells (HMECs). CXCR2/IL8RB is a chemokine receptor of the GPCR superfamily. It has specificity for pro-angiogenic chemokines: IL-8, CXCL1, 2 and 3 (GROα, β and γ), CXCL5 (ENA-78), CXCL6 (GCP2) and CXCL7 (NAP2) whereas CXCR1 binds only to a sub-group of these: GCP2, NAP2 and IL-8. Moreover, the depletion of CXCR2 partially prevents the growth arrest observed during OIS and causes a reduction in the DNA damage response. Conversely ectopic expression of CXCR2 (or its paralogue CXCR1) caused senescence in a manner mainly dependent of p53. Interestingly, the expression of most, if not all, CXCR2 ligands is upregulated during OIS, as is the expression of the chemokine receptor CXCR2 itself.7 These observations suggest that CXCR2 signal- Figure 1. Role of CXCR2 in senescence. The expression of CXCR2 and its ligands is ling could be part of a feedback mechanisms involved upregulated in senescent cells. Activation of NFkB and C/EBPb mediate the transcriptional in reinforcing senescence. Similar conclusions can be upregulation of CXCR2 binding chemokines such as IL-8, GROa and others. Once secreted, drawn from the upregulation of the pair IL6/IL6R CXCR2 ligands can exert multiple paracrine effects, often promoting tumour progression, but are also involved in triggering an innate immune response. In addition, CXCR2 during senescence.7,8 In addition, it suggests that some ligands can also act in a paracrine loop to reinforce senescence, in a process that involves of the effects exerted by other secreted factors during increased production of ROS, which eventually result in augmented DNA damage. senescence might be also mediated by the coordinated upregulation of ligand/receptor pairs. We wondered whether besides receptor that engages multiple pathways once activated.18 CXCR2 all the secreted factors upregulated during senescence, an increase activation by their binding chemokines results in activation of in levels of some of their respective receptors also takes place. For NFκB, MAPK, PI3K and Rac among other signalling cascades. The CXCR2-binding chemokines, their coordinated upregulation with activation of Rac by CXCR2 recruits NAPDH oxidases to produce CXCR2 hints that their more prominent effect would be to reinforce a burst of reactive oxygen species (ROS). This ROS burst has been senescence, as senescent cells would already have upregulated the implicated in the clearing of pathogen infections by macrophages expression of CXCR2, enabling them to respond to the extracellular and also mediates the induction of apoptosis in cancer cells.19 Our chemokines. However, we cannot discard that depending of the cell data suggest that the production of ROS in response to CXCR2 type and basal levels of CXCR2, the secretion of CXCR2 ligands activation is also behind its ability to induce senescence (Fig. 1). could also act by ‘spreading’ senescence to neighbouring normal cells CXCR2 triggers senescence in normal cells in a way at least partially through paracrine signalling. dependent on p53 and concomitant with DNA damage.7 Changes In this regard our experiments using neutralizing antibodies or in Rac1 activity have been linked previously with the induction of recombinant proteins, suggest that the secreted pool of chemokines premature senescence in primary cells.20 The authors suggest that the at least partially mediate the effects of CXCR2 during senescence,7 effects observed upon manipulation of Rac1 levels are mediated by its and these effects are not exclusively due to activation by an intrac- impact on production of ROS. Indeed, increased ROS production is ellular ligand pool as seems to be the case for IL-6.8 Independent able to provoke or sensitise towards DNA damage and contributes to evidence for CXCR2-ligands inducing senescence through paracrine accelerate replicative senescence.21 ROS produced by oncogenic Ras mechanisms can be found in a previous report from Yang et al.17 also are known to mediate the arrest observed during OIS.22 They observed that the addition of recombinant GROa to normal One of the first questions that arose after the observation that ovarian fibroblast results in senescence as analyzed using different CXCR2 signaling mediates senescence in cell culture was to establish markers. The authors suggest that GROa secreted by ovarian epithe- its relevance in vivo. At first instance, a role for CXCR2 signaling lial cancer cells could cause senescence of stromal fibroblasts, and in reinforcing senescence seemed counterintuitive when analyzed these senescent fibroblast themselves support tumorigenesis17 in line together with the established roles for IL-8 and GROa as positive with similar observations from Campisi’s group.9 mediators in tumor progression.18,23 Both IL-8 and GROa have The precise nature of the mechanism by which CXCR2 mediates well-known pro-tumorigenic activities and specifically IL-8 has been senescence still remains unsolved. CXCR2 is a G-protein coupled identified as a mediator of the pro-tumorigenic effects of Ras.24 www.landesbioscience.com
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of the more important transcription factors associated with aging.31 Meta-analysis of those profiles have suggested however, that the group of genes controlled by NFκB relevant for their effects during aging are not cytokines and chemokines, but genes involved in mitotic checkpoint, cell cycle progression and chromatin remodeling. Our preliminary experiments suggest that interfering globally with NFκB activity, despite blocking the SASP response during senescence, does not necessarily result in increased cell numbers. A possible explanation is that in the context of Ras activation, the inhibition of NFκB might result in increased apoptosis,32 therefore masking its putative effects on senescence bypass. In view of the recent results which link secreted factors and senescence,13 we wonder whether there still remain unidentified factors that could influence senescence in a paracrine or autocrine fashion. Their identification is a clear priority as secreted factors that regulate senescence and by extension cellular lifespan could have multiple applications. Most if not all biopharmaceuticals are secreted molecules, and many of the small molecule drugs target cellular receptors. Therefore, the possibility to regulate those targets by intervening pharmacologically, to either boost or prevent senescence seems attractive. These interventions could happen on pathological manifestations of senescence in vivo, such as preneoplastic lesions or other senescence-associated pathologies. In addition, if compounds are identified (for example CXCR2 inhibitors) that can prolong the lifespan of primary cells in vitro, this would have added interest, as they could be employed for improving the in vitro growth of cells used for biotechnological applications or regenerative medicine. In summary, beyond the interest in clarifying the relative contribution of CXCR2 signaling to tumor progression in vivo, our study joins recent work from several laboratories to highlight the various pleiotropic actions exerted by factors secreted by senescent cells.
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IL-8 and GROa have been linked with promoting tumor growth, increasing angiogenesis, vascularization, and favoring metastasis. Evidence for these pro-tumorigenic actions is supported by data obtained in experimental systems and by the analysis of human tumors. Our work, analyzing several models in which senescence is associated with tumor progression, suggests that beyond its pro-tumorigenic activities CXCR2 expression is found linked to senescence both in experimental and therapeutic systems in vivo. The following need to be taken into consideration to be able to integrate these observations: Firstly, senescence mediated via CXCR2 signaling is dependent on the genetic context. We observed that the ectopic expression of CXCR2 causes senescence in MEFs of different genotypes, but not in MEFs derived from mice lacking p53.7 These observations are consistent with the ability of overexpresed CXCR2 to transform immortalized mouse fibroblasts.25 Although paradoxical, these context-dependent effects are a recurrent theme in cancer biology. The number of genes that can exert opposing actions on transformation depending of the context such as Ras,26 KLF4,27 HMGA28 or TGFb29 is steadily increasing and probably reflects that oncogene signaling is associated with complex regulatory mechanisms to prevent aberrant cell proliferation. The second consideration is that at the same time that chemokines such as IL-8 or GROa signal in an autocrine manner to reinforce senescence, their paracrine action on different cell types could be protumorigenic. Most of the evidences for pro-tumorigenic effects of IL-8 or GROa, refer to paracrine effects exerted over other cell types to promote vascularization, angiogenesis or metastasis. It is not very difficult to imagine that different cell types can be wired differently to respond to the same signals (in this case CXCR2 ligands). In addition, the possible activation or production of mediators required for specific pathways downstream of CXCR2 could direct the response towards a particular cell fate. Therefore, an unexpected conclusion of our studies is that CXCR2 signaling might act as a double-edged sword either promoting or inhibiting cancer progression. This should be taken into account when considering therapeutic approaches. Depending on the status, or stage of the tumors the effects of inhibitory or agonistic therapy targeting CXCR2 could be diverse and perhaps opposite to the expectations. The use of animal models will contribute to clarifying the relevance of senescence mediated by CXCR2 in different pathological settings. As components of the ‘CXCR2 signaling system’ are found upregulated during senescence and signaling via CXCR2 can induce senescence, the idea of a self-activating loop reinforcing senescence takes strength. Indeed, the mechanism that controls the strong pro-inflammatory cytokine response during viral infection and inflammation is so similar that it could have been borrowed by the senescent response. During inflammation, cytokine signaling causes the activation of a cascade of transcription factors that synergize in a feedback loop to enhance the production of these same cytokines and additional ones.30 During senescence, this mechanism might operate with NFκB and C/EBP cooperating to regulate the expression of IL-8 and additional CXCR2 ligands. Although these pathways are not necessarily linear, evidence from Daniel Peeper’s work suggest that indeed C/EBPb activation has a pivotal role in controlling senescence.8 Dissecting the impact of NFκB in senescence is not so straightforward as NFκB also acts as an anti-apoptotic factor. Analysis of transcriptional profiles have showed that NFκB is one 2958
Acknowledgements
We thank CSC Photo Services for their help with the preparation of the figure. The Medical Research Council and grants from Cancer Research UK and the Association for International Cancer Research fund the research in our laboratory. Ana Banito is funded by a fellowship from Fundação para a Ciência ea Tecnologia, FCT. References 1. Hayflick L. The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res 1965; 37:614-36. 2. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007; 8:729-40. 3. Mason DX, Jackson TJ, Lin AW. Molecular signature of oncogenic ras-induced senescence. Oncogene 2004; 23:9238-46. 4. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD. Microarray analysis of replicative senescence. Curr Biol 1999; 9:939-45. 5. Narita M, Nunez S, Heard E, Lin AW, Hearn SA, Spector DL, et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003; 113:703-16. 6. Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K, et al. Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol 2006; 1291-7. 7. Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell 2008; 133:1006-18. 8. Kuilman T, Michaloglou C, Vredeveld LC, Douma S, van Doorn R, Desmet CJ, et al. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 2008; 133:1019-31. 9. Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA 2001; 98:12072-7. 10. Parrinello S, Coppe JP, Krtolica A, Campisi J. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation. J Cell Sci 2005; 118:485-96.
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11. Coppé JP, Kauser K, Campisi J, Beauséjour CM. Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J Biol Chem 2006; 29568-74. 12. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 2007; 445:656-60. 13. Cichowski K, Hahn WC. Unexpected pieces to the senescence puzzle. Cell 2008; 133:958-61. 14. Kortlever RM, Higgins PJ, Bernards R. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence. Nat Cell Biol 2006; 8:877-84. 15. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008; 132:363-74. 16. Gomis RR, Alarcon C, Nadal C, Van Poznak C, Massague J. C/EBPbeta at the core of the TGFbeta cytostatic response and its evasion in metastatic breast cancer cells. Cancer Cell 2006; 10:203-14. 17. Yang G, Rosen DG, Zhang Z, Bast RC, Mills GB, Colacino JA, et al. The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis. Proc Natl Acad Sci USA 2006; 16472-7. 18. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004; 4:540-50. 19. Zhao M, Wimmer A, Trieu K, Discipio RG, Schraufstatter IU. Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2. J Biol Chem 2004; 279:49259-67. 20. Debidda M, Williams DA, Zheng Y. Rac1 GTPase regulates cell genomic stability and senescence. J Biol Chem 2006; 281:38519-28. 21. Passos JF, Saretzki G, Ahmed S, Nelson G, Richter T, Peters H, et al. Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence. PLoS Biol 2007; 5:110. 22. Lee AC, Fenster BE, Ito H, Takeda K, Bae NS, Hirai T, et al. Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J Biol Chem 1999; 274:7936-40. 23. Yuan A, Chen JJ, Yao PL, Yang PC. The role of interleukin-8 in cancer cells and microenvironment interaction. Front Biosci 2005; 10:853-65. 24. Sparmann A, Bar-Sagi D. Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis. Cancer Cell 2004; 6:447-58. 25. Burger M, Burger JA, Hoch RC, Oades Z, Takamori H, Schraufstatter IU. Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi’s sarcoma herpesvirus-G protein-coupled receptor. J Immunol 1999; 163:2017-22. 26. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997; 88:593-602. 27. Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol 2005; 7:1074-82. 28. Narita M, Narita M, Krizhanovsky V, Nunez S, Chicas A, Hearn SA, et al. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation. Cell 2006; 126:503-14. 29. Siegel PM, Massague J. Cytostatic and apoptotic actions of TGFbeta in homeostasis and cancer. Nat Rev Cancer 2003; 3:807-21. 30. Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin-8 gene expression. J Leukoc Biol 2002; 72:847-55. 31. Adler AS, Sinha S, Kawahara TL, Zhang JY, Segal E, Chang HY. Motif module map reveals enforcement of aging by continual NFkappaB activity. Genes Dev 2007; 21:3244-57. 32. Mayo MW, Wang CY, Cogswell PC, Rogers-Graham KS, Lowe SW, Der CJ, et al. Requirement of NFkappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 1997; 278:1812-5.
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Role of CXCR2 on senescence
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