Marco Demaria

An Essential Role for Senescent Cells in Optimal Wound Healing through Secretion of PDGF-AA

Cellular senescence suppresses cancer by halting the growth of premalignant cells, yet the accumulation of senescent cells is thought to drive age-related pathology through a senescence-associated secretory phenotype (SASP), the function of which is unclear. To understand the physiological role(s) of the complex senescent phenotype, we generated a mouse model in which senescent cells can be visualized and eliminated in living animals. We show that senescent fibroblasts and endothelial cells appear very early in response to a cutaneous wound, where they accelerate wound closure by inducing myofibroblast differentiation through the secretion of platelet-derived growth factor AA (PDGF-AA). In two mouse models, topical treatment of senescence-free wounds with recombinant PDGF-AA rescued the delayed wound closure and lack of myofibroblast differentiation. These findings define a beneficial role for the SASP in tissue repair and help to explain why the SASP evolved.

MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.

Lamin B1 loss is a senescence-associated biomarker

This study rigorously defines lamin B1 loss as a marker of senescence in response to all classic signals of senescence, including DNA damage, oncogene activation, and replicative exhaustion. This decline is induced by activation of either the p53 or the pRb pathway and occurs in vivo in response to a senescence-inducing dose of radiation.

The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

Transcriptional control of cell senescence Senescent cells that have stopped proliferating secrete molecules that influence the cells around them. Prevention of this senescence-activated secretory phenotype seems to slow organismal aging. Kang et al. explored the regulatory process behind cell senescence and found that DNA damage led to stabilization of the transcription factor GATA4 (see the Perspective by Cassidy and Narita). Increased activity of GATA4 in senescent cells stimulated genes encoding secreted factors. GATA4 also accumulates in the brains of aging mice or humans. Science, this issue 10.1126/science.aaa5612; see also p. 1448 The transcription factor GATA4 promotes cell senescence. [Also see Perspective by Cassidy and Narita] INTRODUCTION Cellular senescence is a program of arrested proliferation and altered gene expression triggered by many stresses. Although it is a potent tumor-suppressive mechanism, senescence has been implicated in several pathological processes including aging, age-associated diseases, and (counterintuitively) tumorigenesis. One potential mechanism through which senescent cells exert such pleiotropic effects is the secretion of proinflammatory cytokines, chemokines, growth factors, and proteases, termed the senescence-associated secretory phenotype (SASP), which affects senescent cells and their microenvironment. The mechanism by which the SASP is initiated and maintained is not well characterized beyond the classical regulators of inflammation, including the transcription factors NF-κB and C/EBPβ. RATIONALE In senescence growth arrest, two core senescence-regulating pathways, p53 and p16INK4a/Rb, play a critical role. By contrast, the SASP does not depend on either p53 or p16INK4a, which suggests the existence of an independent senescence regulatory network that controls the SASP. Having observed high levels of induction of microRNA miR-146a during induced senescence in human fibroblasts, we developed a green fluorescent protein–tagged senescence reporter based on a miR-146a promoter fragment. This reporter responded to senescence-inducing stimuli, including replicative exhaustion, DNA damage, and oncogenic RAS activation—all of which activate the SASP. This system allowed us to identify additional regulators of senescence and the SASP. RESULTS Through miR-146a promoter analysis, we mapped the critical region for senescence-induced activity and identified the transcriptional regulator responsible for this regulation, GATA4, previously known as a regulator of embryonic development. Ectopic expression of GATA4 induced senescence, whereas disruption of GATA4 suppressed it, thus establishing GATA4 as a senescence regulator. GATA4 protein abundance, but not mRNA, increased during sene1scence, primarily as a result of increased protein stability. Under normal conditions, GATA4 binds the p62 autophagy adaptor and is degraded by selective autophagy. Upon senescence induction, however, this selective autophagy was suppressed through decreased interaction between GATA4 and p62, thereby stabilizing GATA4. GATA4 in turn induced TRAF3IP2 (tumor necrosis factor receptor–associated factor interacting protein 2) and IL1A (interleukin 1A), which activate NF-κB to initiate and maintain the SASP, thus facilitating senescence. GATA4 pathway activation depends on the key DNA damage response (DDR) kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3–related), as does senescence-associated activation of p53 and p16INK4a. However, the GATA4 pathway is independent of p53 and p16INK4a. Finally, GATA4 protein accumulated in multiple tissues in mice treated with senescence-inducing stimuli and during normal mouse and human aging, including many cell types in the brain; these findings raise the possibility that the GATA4 pathway drives age-dependent inflammation. CONCLUSION Our results indicate that GATA4 connects autophagy and the DDR to senescence and inflammation through TRAF3IP2 and IL1A activation of NF-κB. These findings establish GATA4 as a key switch activated by the DDR to regulate senescence, independently of p53 and p16INK4a. Our in vivo data indicate a potential role of GATA4 during aging and its associated inflammation. Because accumulation of senescent cells is thought to promote aging and aging-associated diseases through the resulting inflammatory response, inhibiting the GATA4 pathway may provide an avenue for therapeutic intervention. GATA4 functions as a key switch in the senescence regulatory network to activate the SASP. The nonsenescent state is maintained by inhibitory barriers that prevent cell cycle arrest and inflammation. Upon senescence-inducing signals, ATM and ATR relieve inhibition of the p53 and p16INK4a pathways to induce growth arrest and also block p62-dependent autophagic degradation of GATA4, resulting in NF-κB activation and SASP induction. Cellular senescence is a terminal stress-activated program controlled by the p53 and p16INK4a tumor suppressor proteins. A striking feature of senescence is the senescence-associated secretory phenotype (SASP), a pro-inflammatory response linked to tumor promotion and aging. We have identified the transcription factor GATA4 as a senescence and SASP regulator. GATA4 is stabilized in cells undergoing senescence and is required for the SASP. Normally, GATA4 is degraded by p62-mediated selective autophagy, but this regulation is suppressed during senescence, thereby stabilizing GATA4. GATA4 in turn activates the transcription factor NF-κB to initiate the SASP and facilitate senescence. GATA4 activation depends on the DNA damage response regulators ATM and ATR, but not on p53 or p16INK4a. GATA4 accumulates in multiple tissues, including the aging brain, and could contribute to aging and its associated inflammation.

Constitutively active Stat3 enhances neu-mediated migration and metastasis in mammary tumors via upregulation of Cten.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knock-in mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumor-derived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast.

Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment

Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability. Senescent chondrocytes are found in cartilage tissue isolated from patients undergoing joint replacement surgery, yet their role in disease pathogenesis is unknown. To test the idea that SnCs might play a causative role in OA, we used the p16-3MR transgenic mouse, which harbors a p16INK4a (Cdkn2a) promoter driving the expression of a fusion protein containing synthetic Renilla luciferase and monomeric red fluorescent protein domains, as well as a truncated form of herpes simplex virus 1 thymidine kinase (HSV-TK). This mouse strain allowed us to selectively follow and remove SnCs after anterior cruciate ligament transection (ACLT). We found that SnCs accumulated in the articular cartilage and synovium after ACLT, and selective elimination of these cells attenuated the development of post-traumatic OA, reduced pain and increased cartilage development. Intra-articular injection of a senolytic molecule that selectively killed SnCs validated these results in transgenic, non-transgenic and aged mice. Selective removal of the SnCs from in vitro cultures of chondrocytes isolated from patients with OA undergoing total knee replacement decreased expression of senescent and inflammatory markers while also increasing expression of cartilage tissue extracellular matrix proteins. Collectively, these findings support the use of SnCs as a therapeutic target for treating degenerative joint disease.

Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse

Cellular senescence suppresses cancer by irreversibly arresting cell proliferation. Senescent cells acquire a proinflammatory senescence-associated secretory phenotype. Many genotoxic chemotherapies target proliferating cells nonspecifically, often with adverse reactions. In accord with prior work, we show that several chemotherapeutic drugs induce senescence of primary murine and human cells. Using a transgenic mouse that permits tracking and eliminating senescent cells, we show that therapy-induced senescent (TIS) cells persist and contribute to local and systemic inflammation. Eliminating TIS cells reduced several short- and long-term effects of the drugs, including bone marrow suppression, cardiac dysfunction, cancer recurrence, and physical activity and strength. Consistent with our findings in mice, the risk of chemotherapy-induced fatigue was significantly greater in humans with increased expression of a senescence marker in T cells prior to chemotherapy. These findings suggest that senescent cells can cause certain chemotherapy side effects, providing a new target to reduce the toxicity of anticancer treatments. SIGNIFICANCE Many genotoxic chemotherapies have debilitating side effects and also induce cellular senescence in normal tissues. The senescent cells remain chronically present where they can promote local and systemic inflammation that causes or exacerbates many side effects of the chemotherapy. Cancer Discov; 7(2); 165-76. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

Glucocorticoids suppress selected components of the senescence-associated secretory phenotype.

Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors, and proteases that can alter the tissue microenvironment and may promote age‐related pathology. To identify small molecules that suppress the senescence‐associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are approved for human use. Among the promising library constituents was the glucocorticoid corticosterone. Both corticosterone and the related glucocorticoid cortisol decreased the production and secretion of selected SASP components, including several pro‐inflammatory cytokines. Importantly, the glucocorticoids suppressed the SASP without reverting the tumor suppressive growth arrest and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Suppression of the prototypical SASP component IL‐6 required the glucocorticoid receptor, which, in the presence of ligand, inhibited IL‐1α signaling and NF‐κB transactivation activity. Accordingly, co‐treatments combining glucocorticoids with the glucocorticoid antagonist RU‐486 or recombinant IL‐1α efficiently reestablished NF‐κB transcriptional activity and IL‐6 secretion. Our findings demonstrate feasibility of screening for compounds that inhibit the effects of senescent cells. They further show that glucocorticoids inhibit selected components of the SASP and suggest that corticosterone and cortisol, two FDA‐approved drugs, might exert their effects in part by suppressing senescence‐associated inflammation.

Environmental stress, ageing and glial cell senescence: a novel mechanistic link to Parkinson’s disease?

Exposure to environmental toxins is associated with a variety of age‐related diseases including cancer and neurodegeneration. For example, in Parkinsons disease (PD), chronic environmental exposure to certain toxins has been linked to the age‐related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti‐cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence‐associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age‐related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder.

Senescent Cells and Their Secretory Phenotype as Targets for Cancer Therapy

Cancer is a devastating disease that increases exponentially with age. Cancer arises from cells that proliferate in an unregulated manner, an attribute that is countered by cellular senescence. Cellular senescence is a potent tumor-suppressive process that halts the proliferation, essentially irreversibly, of cells at risk for malignant transformation. A number of anti-cancer drugs have emerged that induce tumor cells to undergo cellular senescence. However, although a senescence response can halt the proliferation of cancer cells, the presence of senescent cells in tissues has been associated with age-related diseases, including, ironically, late-life cancer. Thus, anti-cancer therapies that can induce senescence might also drive aging phenotypes and age-related pathology. The deleterious effects of senescent cells most likely derive from their senescence-associated secretory phenotype or SASP. The SASP entails the secretion of numerous inflammatory cytokines, growth factors and proteases that can render the tissue microenvironment favorable for tumor growth. Here, we discuss the beneficial and detrimental effects of inducing cellular senescence, and propose strategies for targeting senescent cells as a means to fight cancer.