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Senescence: a new weapon for cancer therapy
Review
Senescence: a new weapon for cancer therapy Juan Carlos Acosta and Jesu´s Gil Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, UK
Senescence is a stable cell cycle arrest that can be activated by oncogenic signaling and manifests with changes in cellular organization and gene expression, such as the induction of a complex secretome. Importantly, senescence limits tumor progression and determines the outcome of conventional anticancer therapies. In recent years, therapeutic approaches such as p53 reactivation, inhibition of c-MYC in addicted tumors or treatment with cyclin-dependent kinase (CDK) inhibitors have proven effective by invoking a senescence response. The possibility of using prosenescence therapies for cancer treatment has provoked considerable interest. We propose that the senescence secretome can be a source of novel targets for prosenescence therapies, as it has tumor suppressive actions. Overall, tailored prosenescence therapies have the potential to be used for treating cancer and other pathologies. Replicative senescence: an arresting cell state Senescence is a stable cell cycle arrest induced at the end of the cellular lifespan or in response to different stresses. What we refer to today as replicative senescence was first described by Hayflick and Moorhead [1], who challenged the existing dogma that normal cells were capable of unlimited proliferation in culture [2]. Although senescent cells remain arrested even when stimulated by growth factors, these cells are metabolically active. Senescent cells also display senescence-associated b-galactosidase (SA-bGal) activity, as a consequence of an increase in lysosome number [3], and undergo chromatin remodeling giving rise to the so-called senescence-associated heterochromatin foci (SAHF) [4]. In addition, senescent cells secrete a complex mixture of extracellular matrix and soluble factors, referred to as the senescence-associated secretory phenotype (SASP) or senescence messaging secretome (SMS) [5,6]. Recent studies suggest that manifestations of the senescent phenotype influence the stable cell cycle arrest characteristic of senescent cells [5]. In human cells, replicative senescence is triggered by a combination of two main factors. The first is the activation of a DNA damage response mainly triggered by telomere shortening and uncapping [7]. The second is the derepression of the INK4/ARF (inhibitor of CDK4A/alternative reading frame) locus, which behaves as a sensor for linking stress detection with activation of key tumor suppressor networks [8]. The relative contribution of these pathways Corresponding author: Gil, J. ([email protected]). Keywords: senescence; cancer; therapy; tumor suppressors; secretome; p53; Myc; Ras.
to other types of senescence and how they are engaged are very much dependent on cell type, organism of origin and the specific stimuli activating senescence. Oncogene-induced senescence (OIS) as a barrier for tumor progression Expression of oncogenic RasG12V in normal cells induces a phenotype almost indistinguishable from replicative senescence, termed OIS (Figure 1). Besides OIS, agents causing DNA damage, oxidative stress, chemotherapeutic drugs or even the process of reprogramming to induced pluripotent stem cells can also trigger premature senescence or stress-induced senescence [9]. In 2005, several groups independently identified the presence of cells undergoing OIS in premalignant mouse and human lesions and their conspicuous absence in more advanced tumors [10–14]. Prototypic examples of such lesions are nevi, skin lesions of a benign nature that are precursors to melanomas and that can persist in the skin for years. Human nevi stain positive for SA-b-Gal activity and express high levels of p16INK4a, although in a mosaic pattern. Almost all human nevi bear an activated BRAF or NRAS oncogene, but only after they acquire additional mutations that cancel OIS can they progress to melanoma [12,15]. Senescent cells are present in a wide range of premalignant lesions. Mouse models have shown that oncogenic KRasG12V expression associates with senescence in early stages of lung and pancreatic tumors [14]. In mice harboring an Em-N-Ras transgene [13], most of the animals develop a nonlymphoid neoplasia with prevalent signs of senescence. Conversely, the expression of Em-N-Ras in mouse knockouts for p53 or Suv39h1 causes aggressive T cell lymphomas [13]. Skin papillomas induced by treatment with DMBA and TPA bear activating point mutations in H-Ras [16] and are also enriched in senescent cells [14], in a way that is dependent upon p38 activation by PRAK [17]. Moreover, in a serrate colon cancer model driven by oncogenic K-Ras, hyperplasias with characteristics of senescence, such as elevated p16Ink4a expression and SA-b-Gal activity, are observed [18,19]. Nevertheless, the ability of oncogenic Ras to induce senescence depends on the genetic context and relative expression. Lower levels of Ras in the mammary gland stimulate proliferation and mammary epithelial hyperplasias whereas higher levels of Ras lead to senescence and Ink4a/Arf upregulation [20]. More strikingly, a recent report suggests that activation of K-RasG12D in the pancreas does not trigger senescence but in fact suppresses senescence induced by
0962-8924/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2011.11.006 Trends in Cell Biology, April 2012, Vol. 22, No. 4
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Normal cells Oncogenic alterations Aberrant proliferation
Fail-safe mechanisms Oncogene-induced senescence (OIS) Apoptosis Additional alterations
Cancer cells Conventional therapies
Prosenescence therapies
Therapy-induced senescence (TIS)
Apoptosis TRENDS in Cell Biology
Figure 1. Role of senescence in cancer progression and therapy. Normal cells accumulate oncogenic alterations that trigger an initial phase of aberrant cell proliferation giving rise to preneoplastic lesions. Parallel to this aberrant proliferation, cell-intrinsic fail-safe mechanisms such as oncogene-induced senescence (OIS) and apoptosis are activated. During cancer progression, additional mutations are acquired to override these protective mechanisms, giving rise to full-blown malignancies. Conventional treatments such as chemo- or radiotherapies act by inducing cell death or senescence (termed therapy-induced senescence, TIS). Currently, prosenescence therapies are being explored as an alternative or complement to cancer treatment. Normal cells, gray; preneoplastic cells, yellow; senescent cells, blue; apoptotic cells, purple; cancer cells, red.
inflammation through a mechanism involving Twist-mediated inactivation of p16Ink4a [21]. Other work has suggested that K-RasG12D can only trigger senescence when a detoxifying Nrf2-dependent program commonly activated by oncogenes is disabled [22]. Premalignant lesions caused by oncogenes other than Ras are also enriched in senescent cells. Tissue-specific expression of an oncogenic BRAFV600E mutant results in lung adenomas or melanocytic nevi with increased numbers of senescent cells [23–25]. Deregulated expression of E2F3 in the mouse pituitary gland induces hyperplasias with senescent characteristics [10], whereas c-Myc, usually linked to apoptosis, triggers senescence in murine lymphomas through macrophage-mediated TGF-b secretion [26]. Recent work taking advantage of Bmi / mice has revealed that several leukemic fusion proteins also induce senescence [27]. Inactivation of tumor suppressors also drives senescence in premalignant lesions. The prototypic example is the inactivation of PTEN in the mouse prostate, which induces a phenotype resembling prostate intraepithelial neoplasia (PIN) [11]. PIN lesions have characteristics of senescence, such as positive staining for SA-b-Gal activity 212
and activation of the Arf–p53–p21 axis in mouse cells. Importantly, features of senescence are similarly observed in human PIN [11,28]. As some of the characteristics of senescence induced by Pten loss differ from those of senescence driven by RAS/RAF, the Pandolfi group has renamed this type of senescence Pten loss-induced senescence (PICS) [29]. Specifically, PICS can occur independently of DNA replication and in the absence of DNA damage [29]. Loss of other tumor suppressors, such as NF1 in neurofibromas [30], the von Hippel–Lindau tumor suppressor gene (VHL) in kidney carcinomas [31] or Rb in thyroid cancers [32], also result in premalignant lesions with markers of senescence. Overall, there is very convincing evidence that early oncogenic activation gives rise to premalignant lesions but can, in parallel, activate senescence. In addition, disabling this senescence response is needed for tumor progression. For example, loss of Ink4a/Arf on the serrate colon model induces the appearance of adenocarcinomas [18]. Similarly, nevi lesions induced by oncogenic BRAF expression take months or even years to spontaneously evolve to melanomas, but upon inactivation of Cdkn2a or p53, this progression is markedly accelerated [24,25]. p53 loss in
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Pten / PIN lesions also accelerates the appearance of advanced metastatic prostate cancer [11]. These examples suggest that if mutations that disable senescence occur, OIS can be reversed, giving rise to tumors. Although definitive evidence for the reversibility of senescence in vivo is lacking, acute ablation of Rb or p53 in vitro results in senescence reversal [33,34]. In summary, senescence is not only triggered in preneoplastic lesions but also acts as an effective barrier that needs to be disabled during tumor progression (Figure 1).
Using the Em-myc model of lymphomagenesis, work has started on the senescent program that contributes to the outcome of cancer therapy [39]. This senescent response, termed therapy-induced senescence (TIS), relies on engagement of the p16INK4a and p53 tumor suppressor networks. Genetic models in which either of these networks is inactivated show resistance to cancer treatment, directly linking the ability to mount a robust senescence response with the outcome of chemotherapy. It is possible that key effectors of TIS are frequently mutated in advanced cancers. Therefore, resistance to conventional therapies will often be acquired from the very same mutations driving tumor progression.
Activation of senescence during conventional cancer therapies Targeted cancer therapies have been developed in recent decades and proven effective against specific tumors. The first example was Imatinib, an inhibitor of the BCR–ABL fusion used to treat chronic myeloid leukemia (CML). Since then, many other targeted drugs have proven effective, such as Her2 inhibitors in a subset of breast or lung cancers [35], or BRAF inhibitors for the treatment of melanoma [36]. However, even with this collection of targeted drugs, the most widely used treatments for cancer are still chemoand radiotherapies. The rationale behind their effectiveness is that they cause extensive DNA damage in rapidly dividing cells and thus predominantly target cancer cells. This DNA damage response can induce gross aneuploidies, mitotic catastrophe and apoptosis. Interestingly, senescence has also been identified in cancer cells treated with chemotherapeutic drugs or subjected to ionizing radiation (Figure 1) [37,38].
(a) Telomerase inhibition
(b)
Inducing senescence as an alternative therapy for cancer treatment OIS has a crucial role in preventing cancer progression, as there is an active need to bypass OIS for tumors to evolve from indolent stages to malignant phases. This, together with the relevance of TIS in the outcome of conventional cancer therapies, suggests that prosenescence therapies could be effective in cancer treatment. Therefore, drugs aimed at selectively inducing cellular senescence could represent a promising novel approach for cancer intervention. Supporting this, transgenic mice expressing extra copies of key senescent effectors such as p53 or Ink4/Arf have exhibited extended cancer protection, without unwanted side effects [40]. In addition, several examples have emerged recently of drugs exploiting induction or reinforcement of senescence for cancer treatment. We
Cell cycle control Ras
Myc Terc –/–
(c)
GRN163L
Cdk2 –/– CVT-313
(d)
PICS
VO-OHpic
PD0332991
(e)
Myc addiction
Myc
Pten+/– Pten
Cdk4–/–
Pten–/–
BET
Myc
JQ1
Skp2 –/–
MLN4924
p53 reactivation
Non-functional p53 p53
Nutlin or PRIMA-1
Senescent secretome Immune clearance
Immune system TRENDS in Cell Biology
Figure 2. Prosenescence therapies for cancer treatment. The different types of prosenescence therapies discussed in this review are summarized here. The genes targeted in the therapy are listed at the left of the arrows, and examples of small molecule inhibitors used are listed to the right of the arrows. (a) Inhibition of telomerase. (b) Targeting cell cycle control. (c) Pten loss-induced senescence (PICS) in Pten+/ tumors. (d) Targeting Myc addiction. Inhibition of bromodomain and extraterminal (BET) proteins by JQ1 is used as an indirect way to inhibit Myc activity. (e) Reactivation of p53. Cancer cells, red; senescent cells, blue; apoptotic cells, purple; cells from the immune system, green and white.
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Review review those approaches and their underlying rationale in this section (summarized in Figure 2). Inhibiting telomerase activity One of the hallmarks of tumors is their ability to proliferate beyond the normal replicative lifespan [41,42]. A key factor influencing this limit is telomeric length. Eroded or unprotected telomeres resulting from the progressive shortening caused by cellular division can cause chromosomal fusions and activate a DNA damage signaling cascade [7]. To bypass this limitation, most tumors acquire telomerase activity (around 90%). Consistent with these observations, telomerase-deficient mice engineered by deletion of Terc show reduced cancer susceptibility [43], with the only notable exception being p53 / /Terc / mice, in which cancer incidence increases [44]. Therefore, the inhibition of telomerase activity has been proposed as a valid anticancer approach. Different strategies have been pursued to inhibit telomerase activity in tumors [45], with small molecule enzyme inhibitors (such as GRN163L) being the most promising so far. However, given that inhibition of telomerase can also cause gross aneuploidies or apoptosis, the relative contribution of senescence induction when inhibiting telomerase in tumors is unclear. Modulation of CDK activities Despite the deregulated cell cycle of cancer cells, drugs inhibiting CDKs have had limited success as cancer treatments in general clinical trials [46,47]. However, recent studies suggest that targeting specific cell cycle-dependent kinases or CDK inhibitors (CDKI) in the appropriate genetic context can result in synthetic lethal interactions promoting a tumor-specific prosenescence response with therapeutic benefit [48–50]. One of those studies showed that inhibiting CDK2 in c-Myc-driven tumorigenesis induces senescence. Two different small molecule inhibitors that inhibit Cdk2 (CVT313 and CVT-2584) caused Myc-dependent senescence in MEFs, IMR-90 primary human fibroblasts and even in U937 cancer cells [48]. Similarly, targeting Cdk4 unveiled a synthetic lethal interaction with oncogenic K-RasG12V that has therapeutic potential in lung adenocarcinomas [50]. Genetic inactivation of Cdk4 induces senescence of KRasG12V-driven lung adenomas and results in tumor regression and senescence in more advanced adenocarcinomas. Further proving the therapeutic potential of this interaction, treatment with a selective Cdk4 inhibitor (PD0332991) partially prevented or slowed the appearance of non-small cell lung tumors driven by oncogenic KRasG12V [50]. Targeting cell cycle control in the context of Pten loss also induces a prosenescence response with therapeutic significance. Genetic inhibition of Skp2 synergizes with Pten loss in mouse lymphoma, sarcoma, prostate and adrenal cancer to induce senescence and suppress tumorigenesis [49]. Skp2 regulates expression of the CDKI p27, which is a key target downstream of Skp2 in this synthetic lethal interaction. Experiments in mouse models suggested that targeting Skp2 could trigger senescence in tumors driven by Pten inactivation [49]. To target Skp2 activity, the authors used MLN4924, an inhibitor of 214
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neddylation. Cullin, a protein associated with Skp2, is neddylated and treatment with MLN4924 results in Skp2 inhibition, preventing the formation of tumors in a PC3 human prostate cancer cell xenograft model by inducing senescence that was independent of p53. Activation of a PICS response Another strategy to mount a senescent response in Pten heterozygous tumors was proposed recently [29]. It is based in inhibiting Pten activity in Pten+/ tumors. Although inhibiting a tumor suppressor such as Pten is counterintuitive and could potentially be dangerous, proof-of-principle experiments indicate that it can be therapeutically useful. Previous work demonstrated the contrasting effect of deleting one or both alleles of Pten. Treatment with VO-OHpic, a Pten inhibitor, induces senescence specifically and differentially in Pten +/ tumors with no deleterious effect on surrounding Pten +/+ cells. This senescence response is referred as Pten loss-induced senescence (PICS). As Pten overexpression or PI3K inhibition can also cause senescence [30], it seems that the Pten/PI3K pathway must be very finely tuned and small alterations in either direction could have unwanted consequences. Whether this could be a limitation hampering the therapeutic use of PICS remains to be established, but this highlights how the effectiveness of prosenescence therapies is highly dependent on genetic context. Exploiting tumor addiction to c-Myc Prototypic oncogenes such as RAS or MYC have profound effects on cell cycle control, cell growth and cell metabolism – all the criteria necessary to cause a strong, exploitable addiction in tumors [51,52]. Murine models have shown that tumors can become addicted to c-Myc and suggested c-Myc inhibition as a valid target in cancer therapy. Taking advantage of tet-dependent c-Myc expression, it was reported that switching off c-Myc caused senescence in lymphomas, osteosarcomas or hepatocellular carcinomas (HCCs) [53]. The addiction of tumors to c-Myc expression was also shown by taking advantage of a Myc mutant known as OmoMyc [54]. OmoMyc can homodimerize or heterodimerize with wild-type Myc or Max to form complexes with low DNA binding efficiency. Therefore, OmoMyc behaves as a dominant-negative mutant by sequestering and inhibiting c-Myc. Although in early stage K-Ras-induced lung adenomas the tumor suppressive response observed after c-Myc inactivation is mediated by apoptosis induction, in advanced lung adenocarcinomas OmoMyc caused decreased proliferation accompanied by senescence [55]. In addition, inactivation of c-Myc in lymphomas caused senescence and tumor regression in a manner dependent upon CD4+ T lymphocytes. This suggests that beyond cell-intrinsic effects, the tumor microenvironment, and in particular the immune system, can mediate prosenescent tumor suppression [56]. Despite the validity of exploiting tumor addiction to cMyc for therapy, an important obstacle remains in identifying effective inhibitors for clinical use. In this regard, three recent studies identified inhibition of bromodomain and extraterminal (BET) proteins by small molecules such as JQ1 or I-BET151 as a way to affect c-Myc function
Review
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Box 1. The senescence-associated secretory phenotype (SASP) Senescent cells secrete a complex mixture of secreted and extracellular factors usually referred to as the SASP. It has been also called senescence messaging secretome (SMS). The production by senescent cells of secreted factors was noted more than 20 years ago with the identification of individual factors promoting changes in the extracellular matrix and influencing the cellular microenvironment, such as a-(I) procollagen, fibronectin, collagenase, stromelysin and gelatinaseTIMP-2 [74–78]. Gene expression profiles confirmed that senescent cells secrete factors related to wound healing, inflammatory response or cytokine and chemokine signaling [14,79–81]. This upregulation of secreted proteins is common to cells undergoing replicative senescence or OIS and observed in cells of epithelial, fibroblast or endothelial origin [80]. The SASP is present not only in human cells but also in mouse cells undergoing senescence when cultured in 3% oxygen [6]. Antibody arrays have been used for characterizing the senescent secretome [28,82], confirming its complex nature. The senescence secretome includes extracellular proteases (urokinase-type plasminogen activator, uPA), tPA, matrix components (MMP), growth factors, proinflammatory cytokines (IL-6, IL-1a, IL-1b) or chemokines (IL-8, GROa, MCP-1). Transcription factors such as CEBPb and NF-kB act as master regulators of the senescence secretome [28,81,83,84].
[57,58]. This indirect inhibition of c-Myc is a promising therapeutic strategy for acute myeloid leukemia (AML) [57], mixed-lineage leukemia (MLL) [59] and multiple myeloma (MM) [58], as shown using murine models and primary patient samples. In particular, in MM, treatment with JQ1 results in cell cycle arrest and senescence induction, showing that disrupting addiction to c-Myc can engage a senescent response [58]. Reactivation of the tumor suppressive function of p53 As tumors inactivate the p16/Rb and p53 networks to bypass OIS and thrive, the reactivation of these tumor suppressors could conversely restore senescence and halt tumor progression. Although ineffective in tumors sustaining p53 deletions, drugs that enhance p53 function through MDM2 inhibition (such as nutlin) or that can restore the wild-type activity of p53 mutants (such as PRIMA-1) are currently being tested [60]. The validity of this strategy has been shown in mouse models in which p53 expression can be reactivated. Using a Cre-loxP-based transgene, restoration of p53 activity in tumors was shown to cause their regression. Lymphoma regression is caused by massive apoptosis induction, whereas in sarcomas cell cycle arrest with features of senescence is observed [61]. Using a tetinducible shRNA to conditionally regulate endogenous p53 levels in a mosaic mouse model of HCC driven by HRasG12V expression, even a brief reactivation of p53 was shown to result in tumor regression, accompanied by senescence but negligent apoptosis [62]. Interestingly, the activation of senescence in cancer cells was associated with a strong SASP. Secretion of proinflammatory cytokines by senescent cells initiated an innate immune response responsible for tumor clearance [62]. The senescence secretome as a novel target for prosenescence therapies Senescent cells secrete a complex mixture of extracellular proteins and soluble factors that are referred to as the SMS, for their ability to signal and influence their surrounding environment, or the SASP (Box 1). The
The effects exerted by the senescence secretome on the microenvironment and surrounding cells are diverse and can often be opposing. Senescent fibroblasts can promote tumorigenesis of transformed epithelial cells through a combination of cell–cell interaction and soluble factors [85]. Senescent fibroblasts can also contribute to the protumorigenic behavior of neighboring epithelial cancer cells and can, for example, affect the differentiation of epithelial cells [86], promote angiogenesis through VEGF production [87], induce epithelial-to-mesenchymal transition or promote migration of epithelial cancerous cells [82]. However, the senescence secretome can have tumor suppressive effects. Some factors of the SASP, such as PAI-1, IGFBP7, IL-6 or IL-8, have a role in establishing or reinforcing the cell cycle arrest characteristic of senescence [5]. These factors engage the senescence machinery by several different mechanisms: IL-6 induces p15INK4b [81]; IL-8 increases ROS production and causes DNA damage [28]; PAI-1 increases GSK3b activity and disrupts the nuclear localization of cyclin D1 [88]. In addition, factors of the senescence secretome can signal to the immune system to clear senescent cells [62], therefore also contributing to tumor suppression in a non-cell-autonomous way.
senescence secretome exerts diverse and opposite effects over the microenvironment and on neighboring cells [6]. Although initial interest in studying the SASP was focused on its protumorigenic potential, such as promoting growth, migration or angiogenesis, more recently, its tumor suppressive properties have also been highlighted. Components of the senescence secretome reinforce or implement stable cell cycle arrest and contribute to tumor suppression by signaling to and recruiting the immune system [5] (Figure 3). Secreted factors and their receptors are prototypic druggable molecules. Indeed, most biotherapeutics used in the clinic are soluble factors or antibodies targeting such factors or their receptors. Therefore, it is feasible to manipulate components of the senescence secretome for prosenescence therapies and many of the necessary reagents have already been developed. Members of the senescence secretome contributing to senescence arrest include PAI1, IL-8, IL-6, IGFBP7 and TGF-b [5]. However, some of these factors display opposing effects; they can be tumor suppressive or protumorigenic depending on the context. This is the case for proinflammatory cytokines such as IL-6, IL-8 and GROa, which display multiple protumorigenic activities. As a result, IL-6 and IL-8 are both required mediators of Rasdriven tumorigenesis [63,64]. What switches the senescence secretome from tumor suppressive to protumorigenic is not well understood, although it is thought that the genetic context of the target cells (e.g. whether they have intact Rb and p53 pathways) is a factor. Despite increased knowledge on SASP composition and the existence of recombinant proteins, small molecules and antibodies to target these factors, there are no current examples of prosenescence cancer therapies that exploit components of the senescence secretome, at either the experimental or the preclinical stage. However, precedents suggest that such an approach could succeed. Some of the prosenescence therapies described previously, such as reactivation of p53 in Ras-driven HCC [62] or c-Myc lymphomas treated with chemotherapeutic drugs [26], rely on secreted factors to drive senescence and activate innate 215
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Cancer cells Prosenescence therapies
Cell growth Cell differentiation Angiogenesis EMT Migration
Senescent secretome
Cancer cells
Reinforce growth arrest Immune clearance Immune system
Secretome manipulation TRENDS in Cell Biology
Figure 3. The senescence secretome in therapy: roles, possibilities and problems. Therapies that result in senescence induction (prosenescence therapies) stop tumor progression through cell-intrinsic mechanisms and also induce the production of a senescence secretome. This senescence secretome has tumor suppressive effects (shown in red), such as its contribution to reinforce growth arrest and signaling to the immune system for clearance, but can also exert protumorigenic actions in cancer cells (shown in green). By manipulating the secretome, targeting individual components or using it in tumors or preneoplastic lesions of specific genetic composition, we aim to enhance their tumor suppressive effects, minimizing its protumorigenic actions. Cancer cells, red; senescent cells, blue; cells from the immune system, green and white.
immunity. In addition, factors secreted by senescent cells are able to not only invoke innate immunity to clear tumors during prosenescence therapies [62] but also initiate an immune surveillance of premalignant senescent cells that relies on an adaptive immune response [65]. The best example of components of the senescence secretome used in a prosenescence therapy comes not from a model of cancer but from studying the fibrotic response associated with wound healing [66]. The extracellular matrix protein CCN1 (also known as CYR61) restricts fibrosis induction during wound healing. CCN1 is secreted by senescent cells and binds to integrin a6b1 in target cells. This binding activates the RAC1– NOX1 pathway and results in the production of reactive oxygen species (ROS) and senescence induction [66]. The authors observed that senescent fibroblasts preferentially accumulate in granulation tissues of healing wounds and express antifibrotic genes. However, mice expressing a CCN1 mutant that cannot bind to integrin a6b1 exhibit exacerbated fibrosis and do not accumulate senescent fibroblasts. Interestingly, topical application of recombinant CCN1 on cutaneous wounds reduced fibrosis, increased senescence and improved healing [66]. These results suggest that components of the senescence secretome could be successfully used for prosenescence therapies (Figure 3). 216
Concluding remarks Ever since the identification of senescence as a stable growth arrest with potential implications in aging, its physiological relevance has expanded as we realize that stresses such as oncogenes, ionizing radiation or exposure to chemotherapeutic drugs also activate senescence. The roles of senescence in suppressing progression of premalignant lesions and as a determinant of the outcome of cancer therapies have promoted the idea that enhancing senescence (prosenescence therapies) could be an alternative or a complement to conventional anticancer treatments. As such, proof-of-principle studies using mouse models have shown the therapeutic potential of engaging senescence. In parallel, pioneering studies such as those summarized in this review have shown the feasibility of prosenescence therapies. Indeed, several compounds that could be used in prosenescence therapies (summarized in Table 1) are currently undergoing clinical trials. In recent years, the list of pathologies to which senescence is associated has increased well beyond cancer. Senescence limits fibrotic responses during progression to cirrhosis or in cutaneous wound healing [66,67]. Increased incidence of senescence has been shown in diabetes, atherosclerotic plaques and multiple age-related diseases. Moreover, recent genome-wide association studies (GWAS) have shown that multiple single-nucleotide
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Table 1. Molecules with possible applications in prosenescence therapies Strategy Inhibition of telomerase activity
Drug e.g. GRN163L
Efficacy General, most tumors reactivate telomerase
Inhibition of CDK2
CVT-313, CVT-2584
MYC-driven tumors
Inhibition of CDK4
PD0332991
K-RasG12V-driven tumors
Inhibition of Skp2/ neddylation PTEN inhibition
MLN4924
PTEN-null tumors. Works independently of p53 status PTEN+/ tumors
Inhibition of BET family proteins
JQ1, I-BET151
MDM2 inhibition
e.g. Nutlin, RITA
Tumors ‘addicted’ to MYC. Potentially useful in AML, MM and MLL Tumors with wild-type p53
Mutant p53 reactivation
e.g. PRIMA-1, MIRA-1
Tumors with mutant p53
Treatment with CCN1
Recombinant CCN1
Tested in restricting fibrosis in wound healing
VO-OHpic
polymorphisms (SNPs) at non-coding regions of the INK4/ ARF locus, which encodes for key senescence effectors, are associated with Alzheimer’s disease or susceptibility to atherosclerotic vascular disease (ASVD) [68,69]. A mouse model in which the corresponding syngenic region was deleted showed decreased expression of the Ink4/Arf locus and suggested that ASVD is associated with diminished senescence [70]. Although current evidence falls short of suggesting that re-establishment of senescence could be beneficial for ASVD, perhaps prosenescence therapies could also be useful in treating pathologies other than cancer. However, as a note of caution we should mention that increased senescence in vascular cells has been linked to atherosclerosis [71]. We envision that novel prosenescence therapies will be developed in different ways: through improved knowledge of the molecular pathways controlling senescence; by specifically exploiting cell culture systems to screen for senescence regulators; or through the discovery of drugs effective for cancer treatment without a priori knowledge that they will have prosenescence effects. In particular, given the current knowledge on senescence, we suggest that components of the senescence secretome and drugs targeting the epigenetic control of senescence should be explored to find additional prosenescence therapies. Exploiting senescence as an anticancer therapy can be less intuitive than inducing apoptosis, as one results in cell cycle arrest whereas the other effectively eliminates the affected cells. Although there are clear examples (such as nevi) in which senescent cells remain ‘halted’ for years and decades in vivo without active clearance, it is becoming evident that, in other cases, senescent cells signal through their secretome to be actively eliminated by the immune system. Therefore, a possible advantage of prosenescence therapies is that they could work twofold, by intrinsically stopping cell proliferation and eventually by recruiting the immune system to cause clearance of premalignant cells or regression of tumors (Figure 3). A recent study takes
Limitations Possible side effects in normal tissue homeostasis. Inhibition of telomerase increases cancer incidence in p53-null background Limited success in trials with CDK inhibitors so far Limited success in trials with CDK inhibitors so far Unknown side effects of global inhibition of neddylation Balance of PTEN activity is delicate. Potentially dangerous to inhibit a tumor suppressor such as PTEN Identification of adequate tumors to be targeted Seems to have a good therapeutic window, but could select for p53 mutations No clear mechanism of action or not known if it will be effective in different mutants Efficacy as cancer treatment unknown
References [45]
[48] [50] [49] [29]
[57–59]
[60] [60] [66]
advantage of an ingenious mouse model to prove that eliminating senescent cells can improve age-related phenotypes [72]. Therefore, an alternative to prosenescence therapies could be to enhance the elimination of cells undergoing OIS. However, we first need to determine whether ablating senescent cells could be beneficial for tumor suppression and how this could be achieved. A word of caution must be added as senescence regulators can often behave as double-edged swords, having opposite effects on cell proliferation and tumor growth depending on genetic context. Factors of the senescence secretome such as IL-6 and IL-8 can be either tumor suppressive or protumorigenic, slight changes in Pten activity can make a difference between tumor growth or senescence induction, and oncogenic Ras itself either behaves as a potent oncogene or halts proliferation depending on expression levels, genetic context and other factors. Therefore, prosenescence therapy must be applied very specifically, and will require stratification of patients by tumor type and genotyping. An additional challenge remains in monitoring the engagement of senescence after treatment. Although efforts are being taken to establish a senescence index, similar to the apoptotic or proliferation indexes [73], there is a need to identify more reliable and robust markers of senescence and, ideally, methods to detect senescence compatible with advanced in vivo imaging. Overall, we are just starting to see how prosenescence therapies are joining the arsenal of advanced weaponry available for the fight against cancer, and we anticipate that, spearheaded by improved knowledge on the molecular basis of senescence and the ability to monitor it, novel strategies relying on senescence induction will reach the clinic as potential cancer therapies in the coming years. Acknowledgments Core support from the Medical Research Council (MRC) and grants from MRC Technology, Cancer Research UK and the Association for 217
Review International Cancer Research fund the research in J. Gil’s laboratory. J. Gil is also supported by the EMBO Young Investigator Programme.
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Senescence: a new weapon for cancer therapy Juan Carlos Acosta and Jesu´s Gil Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, UK
Senescence is a stable cell cycle arrest that can be activated by oncogenic signaling and manifests with changes in cellular organization and gene expression, such as the induction of a complex secretome. Importantly, senescence limits tumor progression and determines the outcome of conventional anticancer therapies. In recent years, therapeutic approaches such as p53 reactivation, inhibition of c-MYC in addicted tumors or treatment with cyclin-dependent kinase (CDK) inhibitors have proven effective by invoking a senescence response. The possibility of using prosenescence therapies for cancer treatment has provoked considerable interest. We propose that the senescence secretome can be a source of novel targets for prosenescence therapies, as it has tumor suppressive actions. Overall, tailored prosenescence therapies have the potential to be used for treating cancer and other pathologies. Replicative senescence: an arresting cell state Senescence is a stable cell cycle arrest induced at the end of the cellular lifespan or in response to different stresses. What we refer to today as replicative senescence was first described by Hayflick and Moorhead [1], who challenged the existing dogma that normal cells were capable of unlimited proliferation in culture [2]. Although senescent cells remain arrested even when stimulated by growth factors, these cells are metabolically active. Senescent cells also display senescence-associated b-galactosidase (SA-bGal) activity, as a consequence of an increase in lysosome number [3], and undergo chromatin remodeling giving rise to the so-called senescence-associated heterochromatin foci (SAHF) [4]. In addition, senescent cells secrete a complex mixture of extracellular matrix and soluble factors, referred to as the senescence-associated secretory phenotype (SASP) or senescence messaging secretome (SMS) [5,6]. Recent studies suggest that manifestations of the senescent phenotype influence the stable cell cycle arrest characteristic of senescent cells [5]. In human cells, replicative senescence is triggered by a combination of two main factors. The first is the activation of a DNA damage response mainly triggered by telomere shortening and uncapping [7]. The second is the derepression of the INK4/ARF (inhibitor of CDK4A/alternative reading frame) locus, which behaves as a sensor for linking stress detection with activation of key tumor suppressor networks [8]. The relative contribution of these pathways Corresponding author: Gil, J. ([email protected]). Keywords: senescence; cancer; therapy; tumor suppressors; secretome; p53; Myc; Ras.
to other types of senescence and how they are engaged are very much dependent on cell type, organism of origin and the specific stimuli activating senescence. Oncogene-induced senescence (OIS) as a barrier for tumor progression Expression of oncogenic RasG12V in normal cells induces a phenotype almost indistinguishable from replicative senescence, termed OIS (Figure 1). Besides OIS, agents causing DNA damage, oxidative stress, chemotherapeutic drugs or even the process of reprogramming to induced pluripotent stem cells can also trigger premature senescence or stress-induced senescence [9]. In 2005, several groups independently identified the presence of cells undergoing OIS in premalignant mouse and human lesions and their conspicuous absence in more advanced tumors [10–14]. Prototypic examples of such lesions are nevi, skin lesions of a benign nature that are precursors to melanomas and that can persist in the skin for years. Human nevi stain positive for SA-b-Gal activity and express high levels of p16INK4a, although in a mosaic pattern. Almost all human nevi bear an activated BRAF or NRAS oncogene, but only after they acquire additional mutations that cancel OIS can they progress to melanoma [12,15]. Senescent cells are present in a wide range of premalignant lesions. Mouse models have shown that oncogenic KRasG12V expression associates with senescence in early stages of lung and pancreatic tumors [14]. In mice harboring an Em-N-Ras transgene [13], most of the animals develop a nonlymphoid neoplasia with prevalent signs of senescence. Conversely, the expression of Em-N-Ras in mouse knockouts for p53 or Suv39h1 causes aggressive T cell lymphomas [13]. Skin papillomas induced by treatment with DMBA and TPA bear activating point mutations in H-Ras [16] and are also enriched in senescent cells [14], in a way that is dependent upon p38 activation by PRAK [17]. Moreover, in a serrate colon cancer model driven by oncogenic K-Ras, hyperplasias with characteristics of senescence, such as elevated p16Ink4a expression and SA-b-Gal activity, are observed [18,19]. Nevertheless, the ability of oncogenic Ras to induce senescence depends on the genetic context and relative expression. Lower levels of Ras in the mammary gland stimulate proliferation and mammary epithelial hyperplasias whereas higher levels of Ras lead to senescence and Ink4a/Arf upregulation [20]. More strikingly, a recent report suggests that activation of K-RasG12D in the pancreas does not trigger senescence but in fact suppresses senescence induced by
0962-8924/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2011.11.006 Trends in Cell Biology, April 2012, Vol. 22, No. 4
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Normal cells Oncogenic alterations Aberrant proliferation
Fail-safe mechanisms Oncogene-induced senescence (OIS) Apoptosis Additional alterations
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Figure 1. Role of senescence in cancer progression and therapy. Normal cells accumulate oncogenic alterations that trigger an initial phase of aberrant cell proliferation giving rise to preneoplastic lesions. Parallel to this aberrant proliferation, cell-intrinsic fail-safe mechanisms such as oncogene-induced senescence (OIS) and apoptosis are activated. During cancer progression, additional mutations are acquired to override these protective mechanisms, giving rise to full-blown malignancies. Conventional treatments such as chemo- or radiotherapies act by inducing cell death or senescence (termed therapy-induced senescence, TIS). Currently, prosenescence therapies are being explored as an alternative or complement to cancer treatment. Normal cells, gray; preneoplastic cells, yellow; senescent cells, blue; apoptotic cells, purple; cancer cells, red.
inflammation through a mechanism involving Twist-mediated inactivation of p16Ink4a [21]. Other work has suggested that K-RasG12D can only trigger senescence when a detoxifying Nrf2-dependent program commonly activated by oncogenes is disabled [22]. Premalignant lesions caused by oncogenes other than Ras are also enriched in senescent cells. Tissue-specific expression of an oncogenic BRAFV600E mutant results in lung adenomas or melanocytic nevi with increased numbers of senescent cells [23–25]. Deregulated expression of E2F3 in the mouse pituitary gland induces hyperplasias with senescent characteristics [10], whereas c-Myc, usually linked to apoptosis, triggers senescence in murine lymphomas through macrophage-mediated TGF-b secretion [26]. Recent work taking advantage of Bmi / mice has revealed that several leukemic fusion proteins also induce senescence [27]. Inactivation of tumor suppressors also drives senescence in premalignant lesions. The prototypic example is the inactivation of PTEN in the mouse prostate, which induces a phenotype resembling prostate intraepithelial neoplasia (PIN) [11]. PIN lesions have characteristics of senescence, such as positive staining for SA-b-Gal activity 212
and activation of the Arf–p53–p21 axis in mouse cells. Importantly, features of senescence are similarly observed in human PIN [11,28]. As some of the characteristics of senescence induced by Pten loss differ from those of senescence driven by RAS/RAF, the Pandolfi group has renamed this type of senescence Pten loss-induced senescence (PICS) [29]. Specifically, PICS can occur independently of DNA replication and in the absence of DNA damage [29]. Loss of other tumor suppressors, such as NF1 in neurofibromas [30], the von Hippel–Lindau tumor suppressor gene (VHL) in kidney carcinomas [31] or Rb in thyroid cancers [32], also result in premalignant lesions with markers of senescence. Overall, there is very convincing evidence that early oncogenic activation gives rise to premalignant lesions but can, in parallel, activate senescence. In addition, disabling this senescence response is needed for tumor progression. For example, loss of Ink4a/Arf on the serrate colon model induces the appearance of adenocarcinomas [18]. Similarly, nevi lesions induced by oncogenic BRAF expression take months or even years to spontaneously evolve to melanomas, but upon inactivation of Cdkn2a or p53, this progression is markedly accelerated [24,25]. p53 loss in
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Pten / PIN lesions also accelerates the appearance of advanced metastatic prostate cancer [11]. These examples suggest that if mutations that disable senescence occur, OIS can be reversed, giving rise to tumors. Although definitive evidence for the reversibility of senescence in vivo is lacking, acute ablation of Rb or p53 in vitro results in senescence reversal [33,34]. In summary, senescence is not only triggered in preneoplastic lesions but also acts as an effective barrier that needs to be disabled during tumor progression (Figure 1).
Using the Em-myc model of lymphomagenesis, work has started on the senescent program that contributes to the outcome of cancer therapy [39]. This senescent response, termed therapy-induced senescence (TIS), relies on engagement of the p16INK4a and p53 tumor suppressor networks. Genetic models in which either of these networks is inactivated show resistance to cancer treatment, directly linking the ability to mount a robust senescence response with the outcome of chemotherapy. It is possible that key effectors of TIS are frequently mutated in advanced cancers. Therefore, resistance to conventional therapies will often be acquired from the very same mutations driving tumor progression.
Activation of senescence during conventional cancer therapies Targeted cancer therapies have been developed in recent decades and proven effective against specific tumors. The first example was Imatinib, an inhibitor of the BCR–ABL fusion used to treat chronic myeloid leukemia (CML). Since then, many other targeted drugs have proven effective, such as Her2 inhibitors in a subset of breast or lung cancers [35], or BRAF inhibitors for the treatment of melanoma [36]. However, even with this collection of targeted drugs, the most widely used treatments for cancer are still chemoand radiotherapies. The rationale behind their effectiveness is that they cause extensive DNA damage in rapidly dividing cells and thus predominantly target cancer cells. This DNA damage response can induce gross aneuploidies, mitotic catastrophe and apoptosis. Interestingly, senescence has also been identified in cancer cells treated with chemotherapeutic drugs or subjected to ionizing radiation (Figure 1) [37,38].
(a) Telomerase inhibition
(b)
Inducing senescence as an alternative therapy for cancer treatment OIS has a crucial role in preventing cancer progression, as there is an active need to bypass OIS for tumors to evolve from indolent stages to malignant phases. This, together with the relevance of TIS in the outcome of conventional cancer therapies, suggests that prosenescence therapies could be effective in cancer treatment. Therefore, drugs aimed at selectively inducing cellular senescence could represent a promising novel approach for cancer intervention. Supporting this, transgenic mice expressing extra copies of key senescent effectors such as p53 or Ink4/Arf have exhibited extended cancer protection, without unwanted side effects [40]. In addition, several examples have emerged recently of drugs exploiting induction or reinforcement of senescence for cancer treatment. We
Cell cycle control Ras
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Figure 2. Prosenescence therapies for cancer treatment. The different types of prosenescence therapies discussed in this review are summarized here. The genes targeted in the therapy are listed at the left of the arrows, and examples of small molecule inhibitors used are listed to the right of the arrows. (a) Inhibition of telomerase. (b) Targeting cell cycle control. (c) Pten loss-induced senescence (PICS) in Pten+/ tumors. (d) Targeting Myc addiction. Inhibition of bromodomain and extraterminal (BET) proteins by JQ1 is used as an indirect way to inhibit Myc activity. (e) Reactivation of p53. Cancer cells, red; senescent cells, blue; apoptotic cells, purple; cells from the immune system, green and white.
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Review review those approaches and their underlying rationale in this section (summarized in Figure 2). Inhibiting telomerase activity One of the hallmarks of tumors is their ability to proliferate beyond the normal replicative lifespan [41,42]. A key factor influencing this limit is telomeric length. Eroded or unprotected telomeres resulting from the progressive shortening caused by cellular division can cause chromosomal fusions and activate a DNA damage signaling cascade [7]. To bypass this limitation, most tumors acquire telomerase activity (around 90%). Consistent with these observations, telomerase-deficient mice engineered by deletion of Terc show reduced cancer susceptibility [43], with the only notable exception being p53 / /Terc / mice, in which cancer incidence increases [44]. Therefore, the inhibition of telomerase activity has been proposed as a valid anticancer approach. Different strategies have been pursued to inhibit telomerase activity in tumors [45], with small molecule enzyme inhibitors (such as GRN163L) being the most promising so far. However, given that inhibition of telomerase can also cause gross aneuploidies or apoptosis, the relative contribution of senescence induction when inhibiting telomerase in tumors is unclear. Modulation of CDK activities Despite the deregulated cell cycle of cancer cells, drugs inhibiting CDKs have had limited success as cancer treatments in general clinical trials [46,47]. However, recent studies suggest that targeting specific cell cycle-dependent kinases or CDK inhibitors (CDKI) in the appropriate genetic context can result in synthetic lethal interactions promoting a tumor-specific prosenescence response with therapeutic benefit [48–50]. One of those studies showed that inhibiting CDK2 in c-Myc-driven tumorigenesis induces senescence. Two different small molecule inhibitors that inhibit Cdk2 (CVT313 and CVT-2584) caused Myc-dependent senescence in MEFs, IMR-90 primary human fibroblasts and even in U937 cancer cells [48]. Similarly, targeting Cdk4 unveiled a synthetic lethal interaction with oncogenic K-RasG12V that has therapeutic potential in lung adenocarcinomas [50]. Genetic inactivation of Cdk4 induces senescence of KRasG12V-driven lung adenomas and results in tumor regression and senescence in more advanced adenocarcinomas. Further proving the therapeutic potential of this interaction, treatment with a selective Cdk4 inhibitor (PD0332991) partially prevented or slowed the appearance of non-small cell lung tumors driven by oncogenic KRasG12V [50]. Targeting cell cycle control in the context of Pten loss also induces a prosenescence response with therapeutic significance. Genetic inhibition of Skp2 synergizes with Pten loss in mouse lymphoma, sarcoma, prostate and adrenal cancer to induce senescence and suppress tumorigenesis [49]. Skp2 regulates expression of the CDKI p27, which is a key target downstream of Skp2 in this synthetic lethal interaction. Experiments in mouse models suggested that targeting Skp2 could trigger senescence in tumors driven by Pten inactivation [49]. To target Skp2 activity, the authors used MLN4924, an inhibitor of 214
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neddylation. Cullin, a protein associated with Skp2, is neddylated and treatment with MLN4924 results in Skp2 inhibition, preventing the formation of tumors in a PC3 human prostate cancer cell xenograft model by inducing senescence that was independent of p53. Activation of a PICS response Another strategy to mount a senescent response in Pten heterozygous tumors was proposed recently [29]. It is based in inhibiting Pten activity in Pten+/ tumors. Although inhibiting a tumor suppressor such as Pten is counterintuitive and could potentially be dangerous, proof-of-principle experiments indicate that it can be therapeutically useful. Previous work demonstrated the contrasting effect of deleting one or both alleles of Pten. Treatment with VO-OHpic, a Pten inhibitor, induces senescence specifically and differentially in Pten +/ tumors with no deleterious effect on surrounding Pten +/+ cells. This senescence response is referred as Pten loss-induced senescence (PICS). As Pten overexpression or PI3K inhibition can also cause senescence [30], it seems that the Pten/PI3K pathway must be very finely tuned and small alterations in either direction could have unwanted consequences. Whether this could be a limitation hampering the therapeutic use of PICS remains to be established, but this highlights how the effectiveness of prosenescence therapies is highly dependent on genetic context. Exploiting tumor addiction to c-Myc Prototypic oncogenes such as RAS or MYC have profound effects on cell cycle control, cell growth and cell metabolism – all the criteria necessary to cause a strong, exploitable addiction in tumors [51,52]. Murine models have shown that tumors can become addicted to c-Myc and suggested c-Myc inhibition as a valid target in cancer therapy. Taking advantage of tet-dependent c-Myc expression, it was reported that switching off c-Myc caused senescence in lymphomas, osteosarcomas or hepatocellular carcinomas (HCCs) [53]. The addiction of tumors to c-Myc expression was also shown by taking advantage of a Myc mutant known as OmoMyc [54]. OmoMyc can homodimerize or heterodimerize with wild-type Myc or Max to form complexes with low DNA binding efficiency. Therefore, OmoMyc behaves as a dominant-negative mutant by sequestering and inhibiting c-Myc. Although in early stage K-Ras-induced lung adenomas the tumor suppressive response observed after c-Myc inactivation is mediated by apoptosis induction, in advanced lung adenocarcinomas OmoMyc caused decreased proliferation accompanied by senescence [55]. In addition, inactivation of c-Myc in lymphomas caused senescence and tumor regression in a manner dependent upon CD4+ T lymphocytes. This suggests that beyond cell-intrinsic effects, the tumor microenvironment, and in particular the immune system, can mediate prosenescent tumor suppression [56]. Despite the validity of exploiting tumor addiction to cMyc for therapy, an important obstacle remains in identifying effective inhibitors for clinical use. In this regard, three recent studies identified inhibition of bromodomain and extraterminal (BET) proteins by small molecules such as JQ1 or I-BET151 as a way to affect c-Myc function
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Box 1. The senescence-associated secretory phenotype (SASP) Senescent cells secrete a complex mixture of secreted and extracellular factors usually referred to as the SASP. It has been also called senescence messaging secretome (SMS). The production by senescent cells of secreted factors was noted more than 20 years ago with the identification of individual factors promoting changes in the extracellular matrix and influencing the cellular microenvironment, such as a-(I) procollagen, fibronectin, collagenase, stromelysin and gelatinaseTIMP-2 [74–78]. Gene expression profiles confirmed that senescent cells secrete factors related to wound healing, inflammatory response or cytokine and chemokine signaling [14,79–81]. This upregulation of secreted proteins is common to cells undergoing replicative senescence or OIS and observed in cells of epithelial, fibroblast or endothelial origin [80]. The SASP is present not only in human cells but also in mouse cells undergoing senescence when cultured in 3% oxygen [6]. Antibody arrays have been used for characterizing the senescent secretome [28,82], confirming its complex nature. The senescence secretome includes extracellular proteases (urokinase-type plasminogen activator, uPA), tPA, matrix components (MMP), growth factors, proinflammatory cytokines (IL-6, IL-1a, IL-1b) or chemokines (IL-8, GROa, MCP-1). Transcription factors such as CEBPb and NF-kB act as master regulators of the senescence secretome [28,81,83,84].
[57,58]. This indirect inhibition of c-Myc is a promising therapeutic strategy for acute myeloid leukemia (AML) [57], mixed-lineage leukemia (MLL) [59] and multiple myeloma (MM) [58], as shown using murine models and primary patient samples. In particular, in MM, treatment with JQ1 results in cell cycle arrest and senescence induction, showing that disrupting addiction to c-Myc can engage a senescent response [58]. Reactivation of the tumor suppressive function of p53 As tumors inactivate the p16/Rb and p53 networks to bypass OIS and thrive, the reactivation of these tumor suppressors could conversely restore senescence and halt tumor progression. Although ineffective in tumors sustaining p53 deletions, drugs that enhance p53 function through MDM2 inhibition (such as nutlin) or that can restore the wild-type activity of p53 mutants (such as PRIMA-1) are currently being tested [60]. The validity of this strategy has been shown in mouse models in which p53 expression can be reactivated. Using a Cre-loxP-based transgene, restoration of p53 activity in tumors was shown to cause their regression. Lymphoma regression is caused by massive apoptosis induction, whereas in sarcomas cell cycle arrest with features of senescence is observed [61]. Using a tetinducible shRNA to conditionally regulate endogenous p53 levels in a mosaic mouse model of HCC driven by HRasG12V expression, even a brief reactivation of p53 was shown to result in tumor regression, accompanied by senescence but negligent apoptosis [62]. Interestingly, the activation of senescence in cancer cells was associated with a strong SASP. Secretion of proinflammatory cytokines by senescent cells initiated an innate immune response responsible for tumor clearance [62]. The senescence secretome as a novel target for prosenescence therapies Senescent cells secrete a complex mixture of extracellular proteins and soluble factors that are referred to as the SMS, for their ability to signal and influence their surrounding environment, or the SASP (Box 1). The
The effects exerted by the senescence secretome on the microenvironment and surrounding cells are diverse and can often be opposing. Senescent fibroblasts can promote tumorigenesis of transformed epithelial cells through a combination of cell–cell interaction and soluble factors [85]. Senescent fibroblasts can also contribute to the protumorigenic behavior of neighboring epithelial cancer cells and can, for example, affect the differentiation of epithelial cells [86], promote angiogenesis through VEGF production [87], induce epithelial-to-mesenchymal transition or promote migration of epithelial cancerous cells [82]. However, the senescence secretome can have tumor suppressive effects. Some factors of the SASP, such as PAI-1, IGFBP7, IL-6 or IL-8, have a role in establishing or reinforcing the cell cycle arrest characteristic of senescence [5]. These factors engage the senescence machinery by several different mechanisms: IL-6 induces p15INK4b [81]; IL-8 increases ROS production and causes DNA damage [28]; PAI-1 increases GSK3b activity and disrupts the nuclear localization of cyclin D1 [88]. In addition, factors of the senescence secretome can signal to the immune system to clear senescent cells [62], therefore also contributing to tumor suppression in a non-cell-autonomous way.
senescence secretome exerts diverse and opposite effects over the microenvironment and on neighboring cells [6]. Although initial interest in studying the SASP was focused on its protumorigenic potential, such as promoting growth, migration or angiogenesis, more recently, its tumor suppressive properties have also been highlighted. Components of the senescence secretome reinforce or implement stable cell cycle arrest and contribute to tumor suppression by signaling to and recruiting the immune system [5] (Figure 3). Secreted factors and their receptors are prototypic druggable molecules. Indeed, most biotherapeutics used in the clinic are soluble factors or antibodies targeting such factors or their receptors. Therefore, it is feasible to manipulate components of the senescence secretome for prosenescence therapies and many of the necessary reagents have already been developed. Members of the senescence secretome contributing to senescence arrest include PAI1, IL-8, IL-6, IGFBP7 and TGF-b [5]. However, some of these factors display opposing effects; they can be tumor suppressive or protumorigenic depending on the context. This is the case for proinflammatory cytokines such as IL-6, IL-8 and GROa, which display multiple protumorigenic activities. As a result, IL-6 and IL-8 are both required mediators of Rasdriven tumorigenesis [63,64]. What switches the senescence secretome from tumor suppressive to protumorigenic is not well understood, although it is thought that the genetic context of the target cells (e.g. whether they have intact Rb and p53 pathways) is a factor. Despite increased knowledge on SASP composition and the existence of recombinant proteins, small molecules and antibodies to target these factors, there are no current examples of prosenescence cancer therapies that exploit components of the senescence secretome, at either the experimental or the preclinical stage. However, precedents suggest that such an approach could succeed. Some of the prosenescence therapies described previously, such as reactivation of p53 in Ras-driven HCC [62] or c-Myc lymphomas treated with chemotherapeutic drugs [26], rely on secreted factors to drive senescence and activate innate 215
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Cancer cells Prosenescence therapies
Cell growth Cell differentiation Angiogenesis EMT Migration
Senescent secretome
Cancer cells
Reinforce growth arrest Immune clearance Immune system
Secretome manipulation TRENDS in Cell Biology
Figure 3. The senescence secretome in therapy: roles, possibilities and problems. Therapies that result in senescence induction (prosenescence therapies) stop tumor progression through cell-intrinsic mechanisms and also induce the production of a senescence secretome. This senescence secretome has tumor suppressive effects (shown in red), such as its contribution to reinforce growth arrest and signaling to the immune system for clearance, but can also exert protumorigenic actions in cancer cells (shown in green). By manipulating the secretome, targeting individual components or using it in tumors or preneoplastic lesions of specific genetic composition, we aim to enhance their tumor suppressive effects, minimizing its protumorigenic actions. Cancer cells, red; senescent cells, blue; cells from the immune system, green and white.
immunity. In addition, factors secreted by senescent cells are able to not only invoke innate immunity to clear tumors during prosenescence therapies [62] but also initiate an immune surveillance of premalignant senescent cells that relies on an adaptive immune response [65]. The best example of components of the senescence secretome used in a prosenescence therapy comes not from a model of cancer but from studying the fibrotic response associated with wound healing [66]. The extracellular matrix protein CCN1 (also known as CYR61) restricts fibrosis induction during wound healing. CCN1 is secreted by senescent cells and binds to integrin a6b1 in target cells. This binding activates the RAC1– NOX1 pathway and results in the production of reactive oxygen species (ROS) and senescence induction [66]. The authors observed that senescent fibroblasts preferentially accumulate in granulation tissues of healing wounds and express antifibrotic genes. However, mice expressing a CCN1 mutant that cannot bind to integrin a6b1 exhibit exacerbated fibrosis and do not accumulate senescent fibroblasts. Interestingly, topical application of recombinant CCN1 on cutaneous wounds reduced fibrosis, increased senescence and improved healing [66]. These results suggest that components of the senescence secretome could be successfully used for prosenescence therapies (Figure 3). 216
Concluding remarks Ever since the identification of senescence as a stable growth arrest with potential implications in aging, its physiological relevance has expanded as we realize that stresses such as oncogenes, ionizing radiation or exposure to chemotherapeutic drugs also activate senescence. The roles of senescence in suppressing progression of premalignant lesions and as a determinant of the outcome of cancer therapies have promoted the idea that enhancing senescence (prosenescence therapies) could be an alternative or a complement to conventional anticancer treatments. As such, proof-of-principle studies using mouse models have shown the therapeutic potential of engaging senescence. In parallel, pioneering studies such as those summarized in this review have shown the feasibility of prosenescence therapies. Indeed, several compounds that could be used in prosenescence therapies (summarized in Table 1) are currently undergoing clinical trials. In recent years, the list of pathologies to which senescence is associated has increased well beyond cancer. Senescence limits fibrotic responses during progression to cirrhosis or in cutaneous wound healing [66,67]. Increased incidence of senescence has been shown in diabetes, atherosclerotic plaques and multiple age-related diseases. Moreover, recent genome-wide association studies (GWAS) have shown that multiple single-nucleotide
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Table 1. Molecules with possible applications in prosenescence therapies Strategy Inhibition of telomerase activity
Drug e.g. GRN163L
Efficacy General, most tumors reactivate telomerase
Inhibition of CDK2
CVT-313, CVT-2584
MYC-driven tumors
Inhibition of CDK4
PD0332991
K-RasG12V-driven tumors
Inhibition of Skp2/ neddylation PTEN inhibition
MLN4924
PTEN-null tumors. Works independently of p53 status PTEN+/ tumors
Inhibition of BET family proteins
JQ1, I-BET151
MDM2 inhibition
e.g. Nutlin, RITA
Tumors ‘addicted’ to MYC. Potentially useful in AML, MM and MLL Tumors with wild-type p53
Mutant p53 reactivation
e.g. PRIMA-1, MIRA-1
Tumors with mutant p53
Treatment with CCN1
Recombinant CCN1
Tested in restricting fibrosis in wound healing
VO-OHpic
polymorphisms (SNPs) at non-coding regions of the INK4/ ARF locus, which encodes for key senescence effectors, are associated with Alzheimer’s disease or susceptibility to atherosclerotic vascular disease (ASVD) [68,69]. A mouse model in which the corresponding syngenic region was deleted showed decreased expression of the Ink4/Arf locus and suggested that ASVD is associated with diminished senescence [70]. Although current evidence falls short of suggesting that re-establishment of senescence could be beneficial for ASVD, perhaps prosenescence therapies could also be useful in treating pathologies other than cancer. However, as a note of caution we should mention that increased senescence in vascular cells has been linked to atherosclerosis [71]. We envision that novel prosenescence therapies will be developed in different ways: through improved knowledge of the molecular pathways controlling senescence; by specifically exploiting cell culture systems to screen for senescence regulators; or through the discovery of drugs effective for cancer treatment without a priori knowledge that they will have prosenescence effects. In particular, given the current knowledge on senescence, we suggest that components of the senescence secretome and drugs targeting the epigenetic control of senescence should be explored to find additional prosenescence therapies. Exploiting senescence as an anticancer therapy can be less intuitive than inducing apoptosis, as one results in cell cycle arrest whereas the other effectively eliminates the affected cells. Although there are clear examples (such as nevi) in which senescent cells remain ‘halted’ for years and decades in vivo without active clearance, it is becoming evident that, in other cases, senescent cells signal through their secretome to be actively eliminated by the immune system. Therefore, a possible advantage of prosenescence therapies is that they could work twofold, by intrinsically stopping cell proliferation and eventually by recruiting the immune system to cause clearance of premalignant cells or regression of tumors (Figure 3). A recent study takes
Limitations Possible side effects in normal tissue homeostasis. Inhibition of telomerase increases cancer incidence in p53-null background Limited success in trials with CDK inhibitors so far Limited success in trials with CDK inhibitors so far Unknown side effects of global inhibition of neddylation Balance of PTEN activity is delicate. Potentially dangerous to inhibit a tumor suppressor such as PTEN Identification of adequate tumors to be targeted Seems to have a good therapeutic window, but could select for p53 mutations No clear mechanism of action or not known if it will be effective in different mutants Efficacy as cancer treatment unknown
References [45]
[48] [50] [49] [29]
[57–59]
[60] [60] [66]
advantage of an ingenious mouse model to prove that eliminating senescent cells can improve age-related phenotypes [72]. Therefore, an alternative to prosenescence therapies could be to enhance the elimination of cells undergoing OIS. However, we first need to determine whether ablating senescent cells could be beneficial for tumor suppression and how this could be achieved. A word of caution must be added as senescence regulators can often behave as double-edged swords, having opposite effects on cell proliferation and tumor growth depending on genetic context. Factors of the senescence secretome such as IL-6 and IL-8 can be either tumor suppressive or protumorigenic, slight changes in Pten activity can make a difference between tumor growth or senescence induction, and oncogenic Ras itself either behaves as a potent oncogene or halts proliferation depending on expression levels, genetic context and other factors. Therefore, prosenescence therapy must be applied very specifically, and will require stratification of patients by tumor type and genotyping. An additional challenge remains in monitoring the engagement of senescence after treatment. Although efforts are being taken to establish a senescence index, similar to the apoptotic or proliferation indexes [73], there is a need to identify more reliable and robust markers of senescence and, ideally, methods to detect senescence compatible with advanced in vivo imaging. Overall, we are just starting to see how prosenescence therapies are joining the arsenal of advanced weaponry available for the fight against cancer, and we anticipate that, spearheaded by improved knowledge on the molecular basis of senescence and the ability to monitor it, novel strategies relying on senescence induction will reach the clinic as potential cancer therapies in the coming years. Acknowledgments Core support from the Medical Research Council (MRC) and grants from MRC Technology, Cancer Research UK and the Association for 217
Review International Cancer Research fund the research in J. Gil’s laboratory. J. Gil is also supported by the EMBO Young Investigator Programme.
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