Senescence,Wound Healing,and Cancer: the PAI-1 Connection

Prolonged propagation of primary diploid fibroblasts in culture activates an ageing process known as replicative senescence, which is considered to provide a barrier against oncogenic transformation. Remarkably, both cell autonomous tumor-suppressive and cell non-autonomous tumor-promoting effects of senescent cells have been reported. Recently, we described that the p53 target gene plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of replicative senescence. PAI-1 antagonizes the protease urokinase-type plasminogen activator (uPA). Both are secreted factors and involved in heterotypic signaling processes such as wound healing, angiogenesis and metastasis. Both uPA and PAI-1 are expressed in senescent cells and their relative abundance controls proliferation downstream of p53. Here, we present data that the effects of PAI- 1 and uPA in the senescence response are not strictly cell autonomous. We discuss these findings in the context of the emerging roles of PAI-1 and uPA in heterotypic cellular signaling in senescence, wound healing and metastasis.     

Stem cells, senescence, neosis and self-renewal in cancer

We describe the basic tenets of the current concepts of cancer biology, and review the recent advances on the suppressor role of senescence in tumor growth and the breakdown of this barrier during the origin of tumor growth. Senescence phenotype can be induced by (1) telomere attrition-induced senescence at the end of the cellular mitotic life span (MLS*) and (2) also by replication history-independent, accelerated senescence due to inadvertent activation of oncogenes or by exposure of cells to genotoxins. Tumor suppressor genes p53/pRB/p16INK4A and related senescence checkpoints are involved in effecting the onset of senescence. However, senescence as a tumor suppressor mechanism is a leaky process and senescent cells with mutations or epimutations in these genes escape mitotic catastrophe-induced cell death by becoming polyploid cells. These polyploid giant cells, before they die, give rise to several cells with viable genomes via nuclear budding and asymmetric cytokinesis. This mode of cell division has been termed neosis and the immediate neotic offspring the Raju cells. The latter inherit genomic instability and transiently display stem cell properties in that they differentiate into tumor cells and display extended, but, limited MLS, at the end of which they enter senescent phase and can undergo secondary/tertiary neosis to produce the next generation of Raju cells. Neosis is repeated several times during tumor growth in a non-synchronized fashion, is the mode of origin of resistant tumor growth and contributes to tumor cell heterogeneity and continuity. The main event during neosis appears to be the production of mitotically viable daughter genome after epigenetic modulation from the non-viable polyploid genome of neosis mother cell (NMC). This leads to the growth of resistant tumor cells. Since during neosis, spindle checkpoint is not activated, this may give rise to aneuploidy. Thus, tumor cells also are destined to die due to senescence, but may escape senescence due to mutations or epimutations in the senescent checkpoint pathway. A historical review of neosis-like events is presented and implications of neosis in relation to the current dogmas of cancer biology are discussed. Genesis and repetitive re-genesis of Raju cells with transient "stemness" via neosis are of vital importance to the origin and continuous growth of tumors, a process that appears to be common to all types of tumors. We suggest that unlike current anti-mitotic therapy of cancers, anti-neotic therapy would not cause undesirable side effects. We propose a rational hypothesis for the origin and progression of tumors in which neosis plays a major role in the multistep carcinogenesis in different types of cancers. We define cancers as a single disease of uncontrolled neosis due to failure of senescent checkpoint controls.     

Senescent endothelial cells are prone to TNF-α-induced cell death due to expression of FAS receptor

The senescent endothelial cells show various phenotypes which can increase the incidence of inflammatory cardiovascular diseases, but the fundamental basis for such phenotypic changes of senescing cells remains to be elucidated. This study was undertaken to find transmembrane receptors that might be highly expressed in senescent endothelial cells and play a key role in cell death signal transduction. Comparison of mRNA expression in young and senescent human umbilical vein endothelial cells, using a cDNA microarray method, provided a list of transmembrane receptors including the FAS receptor (tumor necrosis factor receptor superfamily member 6) whose expression levels were significantly increased by cellular senescence. Additional studies focused on FAS demonstrated that a high expression of FAS receptor in senescent endothelial cells is responsible for the susceptibility to apoptotic cell death, as the siRNA-mediated suppression of FAS expression in senescent cells prevented the cell death, and overexpression of exogenous FAS in young cells increased cell death. We also verified that FAS expression level was closely associated with the activation of caspase-3 and caspase-9 involved in apoptosis. The senescence-induced transmembrane receptors including the FAS receptor may provide novel therapeutic targets to prevent cardiovascular diseases.     

Pharmacological CDK4/6 inhibition reveals a p53‐dependent senescent state with restricted toxicity

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets.     

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G₂ arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser¹⁵ and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser¹⁵ phosphorylation is associated with enhanced phosphorylation at Ser⁴⁶, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.     

Lessons from senescence: Chromatin maintenance in non-proliferating cells

Cellular senescence is an irreversible proliferation arrest, thought to contribute to tumor suppression, proper wound healing and, perhaps, tissue and organismal aging. Two classical tumor suppressors, p53 and pRB, control cell cycle arrest associated with senescence. Profound molecular changes occur in cells undergoing senescence. At the level of chromatin, for example, senescence associated heterochromatic foci (SAHF) form in some cell types. Chromatin is inherently dynamic and likely needs to be actively maintained to achieve a stable cell phenotype. In proliferating cells chromatin is maintained in conjunction with DNA replication, but how non-proliferating cells maintain chromatin structure is poorly understood. Some histone variants, such as H3.3 and macroH2A increase as cells undergo senescence, suggesting histone variants and their associated chaperones could be important in chromatin structure maintenance in senescent cells. Here, we discuss options available for senescent cells to maintain chromatin structure and the relative contribution of histone variants and chaperones in this process. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.     

Reduced mitochondrial membrane potential and altered responsiveness of a mitochondrial membrane megachannel in p53-induced senescence.

There is accumulating evidence that mitochondrial membrane potential (DeltaPsi(M)) is reduced in aged cells. In addition, a decrease of DeltaPsi(M) has been shown to be an early event in many forms of apoptosis. Here we use a mitochondrial potentiometric dye with in situ laser scanning confocal microscopic (LSCM) imaging to demonstrate that DeltaPsi(M) is dramatically decreased in both the p53-overexpressing, senescent EJ tumor cells and in pre-apoptotic PC12 cells compared to controls. Treatment with cyclosporin A (CSA), which facilitates closure of the mitochondrial permeability transition pore (PTP), was able to reverse the decrease in DeltaPsi(M) in pre-apoptotic PC12 cells but not in the senescent EJ-p53 cells. The capacity to prevent dissipation of DeltaPsi(M) in response to agents that facilitate PTP closure may differentiate cells entering apoptosis from those participating in senescence. Therefore, regulation of the closure of the mitochondrial PTP in the presence of decreased DeltaPsi(M) may be a decisional checkpoint in distinguishing between growth arrest pathways.     

IL-6 Secreted from Senescent Mesenchymal Stem Cells Promotes Proliferation and Migration of Breast Cancer Cells

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.     

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.     

Lamin B1 loss is a senescence-associated biomarker

Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-κB, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.     

Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors

Cells from organisms with renewable tissues can permanently withdraw from the cell cycle in response to diverse stress, including dysfunctional telomeres, DNA damage, strong mitogenic signals, and disrupted chromatin. This response, termed cellular senescence, is controlled by the p53 and RB tumor suppressor proteins and constitutes a potent anticancer mechanism. Nonetheless, senescent cells acquire phenotypic changes that may contribute to aging and certain age-related diseases, including late-life cancer. Thus, the senescence response may be antagonistically pleiotropic, promoting early-life survival by curtailing the development of cancer but eventually limiting longevity as dysfunctional senescent cells accumulate.     

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.     

Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblasts

Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3beta, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3alpha. Co-immunoprecipitation experiments demonstrated that GSK3beta and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3beta in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence.     

To clear,or not to clear (senescent cells)? That is the question

Cellular senescence is an anti‐proliferative program that restricts the propagation of cells subjected to different kinds of stress. Cellular senescence was initially described as a cell‐autonomous tumor suppressor mechanism that triggers an irreversible cell cycle arrest that prevents the proliferation of damaged cells at risk of neoplastic transformation. However, discoveries during the last decade have established that senescent cells can also impact the surrounding tissue microenvironment and the neighboring cells in a non‐cell‐autonomous manner. These non‐cell‐autonomous activities are, in part, mediated by the selective secretion of extracellular matrix degrading enzymes, cytokines, chemokines and immune modulators, which collectively constitute the senescence‐associated secretory phenotype. One of the key functions of the senescence‐associated secretory phenotype is to attract immune cells, which in turn can orchestrate the elimination of senescent cells. Interestingly, the clearance of senescent cells seems to be critical to dictate the net effects of cellular senescence. As a general rule, the successful elimination of senescent cells takes place in processes that are considered beneficial, such as tumor suppression, tissue remodeling and embryonic development, while the chronic accumulation of senescent cells leads to more detrimental consequences, namely, cancer and aging. Nevertheless, exceptions to this rule may exist. Now that cellular senescence is in the spotlight for both anti‐cancer and anti‐aging therapies, understanding the precise underpinnings of senescent cell removal will be essential to exploit cellular senescence to its full potential.