How autophagy both activates and inhibits cellular senescence

Autophagy and cellular senescence are stress responses essential for homeostasis. While recent studies indicate a genetic relationship between autophagy and senescence, whether autophagy acts positively or negatively on senescence is still subject to debate. Although autophagy was originally recognized as a nonspecific lysosomal degradation pathway (general autophagy), increasing evidence supports a selective form of autophagy that mediates the degradation of specific targets (selective autophagy). Our recent study revealed distinctive roles of selective autophagy and general autophagy in the regulation of senescence, at least in part resolving apparently contradictory reports regarding the relationship between these 2 important homeostatic stress responses.     

High glucose suppresses autophagy through the AMPK pathway while it induces autophagy via oxidative stress in chondrocytes

Diabetes (DB) is a risk factor for osteoarthritis progression. High glucose (HG) is one of the key pathological features of DB and has been demonstrated to induce apoptosis and senescence in chondrocytes. Autophagy is an endogenous mechanism that can protect cells against apoptosis and senescence. The effects of HG on autophagy in cells including chondrocytes have been studied; however, the results have been inconsistent. The current study aimed to elucidate the underlying mechanisms, which could be associated with the contrasting outcomes. The present study revealed that HG can induce apoptosis and senescence in chondrocytes, in addition to regulating autophagy dynamically. The present study demonstrated that HG can cause oxidative stress in chondrocytes and suppress the AMPK pathway in a dose-dependent manner. Elimination of oxidative stress by Acetylcysteine, also called N-acetyl cysteine (NAC), downregulated autophagy and alleviated HG-stimulated apoptosis and senescence, while activation of the AMPK signaling pathway by AICAR not only upregulated autophagy but also alleviated HG-stimulated apoptosis and senescence. A combined treatment of NAC and AICAR was superior to treatment with either NAC or AICAR. The study has demonstrated that HG can suppress autophagy through the AMPK pathway and induce autophagy via oxidative stress in chondrocytes.Subject terms: Autophagy, Bone, Endocrine system and metabolic diseases     

Autophagy-dependent senescence in response to DNA damage and chronic apoptotic stress

Autophagy regulates cell survival and cell death upon various cellular stresses, yet the molecular signaling events involved are not well defined. Here, we established the function of a proteolytic Cyclin E fragment (p18-CycE) in DNA damage-induced autophagy, apoptosis, and senescence. p18-CycE was identified in hematopoietic cells undergoing DNA damage-induced apoptosis. In epithelial cells exposed to DNA damage, chronic but not transient expression of p18-CycE leads to higher turnover of LC3 I/II and increased emergence of autophagosomes and autolysosomes. Levels of p18-CycE, which was generated by proteolytic cleavage of endogenous Cyclin E, were greatly increased by chloroquine and correlated with LC 3II conversion. Preventing p18-CycE genesis blocked conversion of LC3 I to LC3 II. Upon DNA damage, cytoplasmic ataxia-telangiectasia-mutated (ATM) was phosphorylated in p18-CycE-expressing cells resulting in sustained activation of the adenosine-mono-phosphate-dependent kinase (AMPK). These lead to sustained activation of mammalian autophagy-initiating kinase ULK1, which was abrogated upon inhibiting ATM and AMPK phosphorylation. Moreover, p18-CycE was degraded via autophagy followed by induction of senescence. Both autophagy and senescence were prevented by inhibiting autophagy, which leads to increased apoptosis in p18-CycE-expressing cells by stabilizing p18-CycE expression. Senescence was further associated with cytoplasmic co-localization and degradation of p18-CycE and Ku70. In brief, chronic p18-CycE expression-induced autophagy leads to clearance of p18-CycE following DNA damage and induction of senescence. Autophagy inhibition stabilized the cytoplasmic p18-CycE-Ku70 complex leading to apoptosis. Thus, our findings define how chronic apoptotic stress and DNA damage initiate autophagy and regulate cell survival through senescence and/or apoptosis.     

Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage

Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.     

Autophagy and senescence: A new insight in selected human diseases

Senescence and autophagy play important roles in homeostasis. Cellular senescence and autophagy commonly cause several degenerative processes, including oxidative stress, DNA damage, telomere shortening, and oncogenic stress; hence, both events are known to be interrelated. Autophagy is well known for its disruptive effect on human diseases, and it is currently proposed to have a direct effect on triggering senescence and quiescence. However, it is yet to be proven whether autophagy has a positive or negative impact on senescence. It is known that elevated levels of autophagy induce cell death, whereas inadequate autophagy can trigger cellular senescence. Both have important roles in human diseases such as aging, renal degeneration, neurodegenerative disorders, and cancer. Therefore, this review aims to highlight the relevance of senescence and autophagy in selected human ailments through a summary of recent findings on the connection and effects of autophagy and senescence in these diseases.     

Autophagy,senescence and tumor dormancy in cancer therapy

The relationships between autophagy and apoptosis have been examined quite extensively and have often been shown to be reciprocally regulated responses to stresses such as exposure of the tumor cells to chemotherapeutic drugs and radiation. However, there is now evidence that autophagy may also play a role in tumor dormancy. Given that tumor dormancy and disease recurrence are poorly understood phenomenon which are nevertheless critical elements of patient morbidity and mortality, this commentary develops the postulate that autophagy and senescence may be related responses that influence the capacity of the tumor cell to maintain a prolonged state of growth arrest that can be succeeded by tumor regrowth and disease recurrence.     

Moderate activation of Wnt/β-catenin signaling promotes the survival of rat nucleus pulposus cells via regulating apoptosis,autophagy, and senescence

This study aimed to investigate the specific role of Wnt/β-catenin signaling in compression-induced apoptosis, autophagy, and senescence in rat nucleus pulposus (NP) cells. Initially, the cells underwent various periods of exposure to 1.0 MPa compression. Wnt/β-catenin signaling associated molecules were assessed in detail, and then 0, 24 and 48 hours exposure periods were selected. The cells were then divided into control, Wnt/β-catenin inhibitor (IWP-2), Wnt/β-catenin activator (LiCl), and β-catenin overexpression groups. After 0, 24, and 48 hours of compression, apoptosis, autophagy, and senescence were evaluated by Western blot analysis and real-time polymerase chain reaction and were visually observed by Hoechst33258, monodansylcadaverine, and SA-β-gal stainings, respectively. Additionally, the regulatory effect of Wnt/β-catenin signaling on cell morphology, viability, cell cycle, death ratio, and ultrastructure was detected to thoroughly evaluate the survival capacity of NP cells. The results established that compression elicited a time-dependent activation of Wnt/β-catenin signaling. The IWP-2 treatment decreased cell survival rate, which corresponded to downregulation of autophagy as well as increases in apoptosis and senescence. LiCl treatment enabled more efficient of cell survival accompanied by increased autophagy and downregulated apoptosis and senescence; however, in contrast to LiCl, overexpression of β-catenin aggravated compression-induced NP cells death. In conclusion, moderate activation of Wnt/β-catenin signaling enables more efficient of NP cells survival via downregulation of apoptosis, senescence, and upregulation of autophagy, and overactivation of Wnt/β-catenin signaling achieved the opposite effect. Treatment strategies that aim to regulate Wnt/β-catenin signaling might be a novel target for improving compression-induced NP cells death and potential treatment of intervertebral disc degeneration.     

The novel phloroglucinol PMT7 kills glycolytic cancer cells by blocking autophagy and sensitizing to nutrient stress

The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.     

Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA–MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.     

Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat

The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as “ER stress”. Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.     

Adaptation to ER stress as a driver of malignancy and resistance to therapy in human melanoma

Primary events in the development of melanoma are gradually being pieced together but a more complete picture of evolution of the disease requires additional understanding of secondary events consequent on initiation of the malignancy. Arguably, the most important driver of secondary events is signals resulting from induction of endoplasmic reticulum (ER) stress for example due to hypoglycaemia and anoxia. This may result in a variety of responses such as apoptosis, autophagy and senescence depending on the initiating event and cell type but most importantly it may result in progression of melanoma due to adaptation and selection of melanoma cells to ER stress. The following reviews what is known about the adaptive responses and how this information may provide new initiatives in treatment of the disease.     

Immunogenic cell death modalities and their impact on cancer treatment

It is still enigmatic under which circumstances cellular demise induces an immune response or rather remains immunologically silent. Moreover, the question remains open under which circumstances apoptotic, autophagic or necrotic cells are immunogenic or tolerogenic. Although apoptosis appears to be morphologically homogenous, recent evidence suggests that the pre-apoptotic surface-exposure of calreticulin may dictate the immune response to tumor cells that succumb to anticancer treatments. Moreover, the release of high-mobility group box 1 (HMGB1) during late apoptosis and secondary necrosis contributes to efficient antigen presentation and cytotoxic T-cell activation because HMGB1 can bind to Toll like receptor 4 on dendritic cells, thereby stimulating optimal antigen processing. Cell death accompanied by autophagy also may facilitate cross priming events. Apoptosis, necrosis and autophagy are closely intertwined processes. Often, cells manifest autophagy before they undergo apoptosis or necrosis, and apoptosis is generally followed by secondary necrosis. Whereas apoptosis and necrosis irreversibly lead to cell death, autophagy can clear cells from stress factors and thus facilitate cellular survival. We surmise that the response to cellular stress like chemotherapy or ionizing irradiation, dictates the immunological response to dying cells and that this immune response in turn determines the clinical outcome of anticancer therapies. The purpose of this review is to summarize recent insights into the immunogenicity of dying tumor cells as a function of the cell death modality.     

The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: oxidative stress and the seesaw of cell survival and death

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.     

Autophagy facilitates oncogene-induced senescence

Oncogenic stress triggers a range of intracellular protective responses including DNA damage checkpoints, senescence and apoptosis, depending on the cell type and the severity of the particular stress. Senescent cells are metabolically viable but are stably arrested. Senescence is a collective phenotype, however, that is comprised of various signaling pathways and effector mechanisms. Thus, to understand and manipulate the senescence phenotype, it is critical to find its effector mechanisms and determine the relationships between them. We have recently found that autophagy is activated upon acute induction of senescence and facilitates another effector mechanism: the senescence associated secretory phenotype (SASP).     

Autophagy induction for the treatment of cancer

Cancer can be viewed in 2 rather distinct ways, namely (i) as a cell-autonomous disease in which malignant cells have escaped control from cell-intrinsic barriers against proliferation and dissemination or (ii) as a systemic disease that involves failing immune control of aberrant cells. Since macroautophagy/autophagy generally increases the fitness of cells as well as their resistance against endogenous or iatrogenic (i.e., relating to illness due to medical intervention) stress, it has been widely proposed that inhibition of autophagy would constitute a valid strategy for sensitizing cancer cells to chemotherapy or radiotherapy. Colliding with this cell-autonomous vision, however, we found that immunosurveillance against transplantable, carcinogen-induced or genetically engineered cancers can be improved by pharmacologically inducing autophagy with caloric restriction mimetics. This positive effect depends on autophagy induction in cancer cells and is mediated by alterations in extracellular ATP metabolism, namely increased release of immunostimulatory ATP and reduced adenosine-dependent recruitment of immunosuppressive regulatory T cells into the tumor bed. The combination of autophagy inducers and chemotherapeutic agents is particularly efficient in reducing cancer growth through the stimulation of CD8⁺ T lymphocyte-dependent anticancer immune responses.     

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.     

Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-β-D-glucose-induced senescence-like growth arrest in human cancer cells

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.