The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.

Summary

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.

Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.

There is little doubt that humans rely on vision as their primary sensory input. However, various studies indicate that audiovisual combinations of data presentation actually enhance the ability of the learner to comprehend the information. We present an example of a musical-biological interface that provides an audible demonstration of SNARE protein function in the process of macroautophagy.

Our laboratory has been investigating for some time the nature of the response of T lymphocytes in autoimmunity in the reactions against self-proteins that result in a number of diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis (RA) and others. T cells recognize peptides generated from proteins that are processed by antigen-presenting cells (APC). The peptides may derive from exogenous proteins or from the normal catabolism of self-proteins. The peptides complexed to major histocompatibility complex (MHC) molecules constitute the chemical entity that is engaged by the antigen-receptor of T cells. An important hypothesis postulates that self-peptides that suffer post-translational modifications in the APC may form neo-antigens that are recognized by the immune system and form the target of autoimmunity. Our interest in citrullination in the context of antigen processing and presentation stemmed from studies suggesting that an immune response to citrullinated self-peptides may be involved in autoimmunity. In a first publication, we found T cells that specifically recognized citrullinated peptides after immunization of inbred mice with standard foreign proteins. We used the small protein hen-egg white lysozyme. These T cells only recognized the citrullinated peptide and not the unmodified one, thus proving that a neo-epitope had been created by this modification. But how this modification took place was not known. Our recent report describes a central role for autophagy in citrullination of peptides by APC.

Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.

Inhibition of the autophagic pathway has recently revealed promising results in increasing pro-death activity of multiple cancer therapeutics. Here, we discuss our findings regarding the autophagy-blocking and anti-neoplastic effects of a synthetic sphingosine analog, FTY720, in mantle cell lymphoma (MCL). We also emphasize how FTY720 enhances the pro-death activity of the fully humanized monoclonal antibody milatuzumab by inhibiting the autophagy-lysosome dependent degradation of its therapeutic target, CD74. Our results provide justification for further evaluation of FTY720 and milatuzumab as a combination therapy for this aggressive B-cell malignancy.

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.

Oxygen (O2), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O2 during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O2 concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O2 concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

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.

During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINRACT and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.

Autophagy allows cells to survive under conditions of nutrient deprivation. We have demonstrated that autophagy inhibitors are synthetically lethal with NFκB inhibitors in B-cell lymphomas because the NFκB pathway promotes survival by increasing glucose import. When NFκB is inhibited in B-cell lymphoma, glucose import decreases and cells become sensitive to perturbations in mitochondrial metabolism and autophagy. Thus, combined inhibition of autophagy and NFκB drives cells into metabolic crisis accelerating cell death.

Autophagy is a fundamental salvage pathway that encapsulates damaged cellular components and delivers them to the lysosome for degradation and recycling. This pathway usually conducts a protective cellular response to nutrient deprivation and various stresses. Tumor cells live with metabolic stress and use autophagy for their survival during tumor progression and metastasis. Genomic instability in tumor cells may result in amplification of crucial gene(s) for autophagy and upregulate the autophagic pathway. We demonstrate that a cancer-associated gene, LAPTM4B, plays an important role in lysosomal functions and is critical for autophagic maturation. Its amplification and overexpression promote autophagy, which renders tumor cells resistant to metabolic and genotoxic stress and results in more rapid tumor growth.

Microtubule-associated protein 1 small form (MAP1S; originally named C19ORF5) was identified as serving as linkers to connect mitochondria with microtubules for trafficking, and to bridge the autophagy machinery with microtubules and mitochondria to affect autophagosomal biogenesis and degradation. We found that MAP1S levels become elevated immediately in response to diethylnitrosamine-induced or genome instability-driven metabolic stress in a murine model of hepatocarcinoma. Elevation of MAP1S enhances autophagy to remove p62-associated aggresomes and dysfunctional organelles that trigger DNA double-strand (DSB) breaks and genome instability. The early accumulation of an unstable genome prior to signs of tumorigenesis suggested that genome instability causes tumorigenesis. After tumorigenesis, tumor development then triggers the activation of autophagy to reduce genome instability in tumor foci. We concluded that an increase in MAP1S levels triggers autophagy in order to suppress genome instability so that both the incidence of diethylnitrosamine-induced hepatocarcinogenesis and malignant progression are suppressed. Thus, a link between MAP1S-enhanced autophagy and suppression of genomic instability and tumorigenesis has been established.

Progressive accumulation of α-synuclein is key to the pathology of many neurodegenerative diseases, including Parkinson disease and dementia with Lewy bodies. Increased intracellular levels of α-synuclein may be caused by enhanced expression or alterations in protein degradation pathways. Here we review our recent study showing that the ubiquitin-proteasome system and the autophagy-lysosomal pathway are differentially involved in α-synuclein's degradation in vivo. We discuss the key findings obtained with our novel in vivo approach and also present a model for the progression of protein aggregation and dysfunctional degradation in Parkinson disease.

Many neurodegenerative conditions have oxidative stress burdens where levels of reactive oxygen species (ROS) exceed the antioxidant capacity of the neuron. ROS can induce wide-ranging damage in a cell and this is prevented by the activation of antioxidant responses including autophagy. Junkinase (JNK) is stimulated by ROS and mediates antioxidant responses via the activation of the transcriptional activators Fos and Jun (AP-1). In recently published work we examined Drosophila mutants with overgrown larval neuromuscular synapses, mutants that also show all the hallmarks of lysosomal storage disease (LSD). We find that we can reverse this synaptic overgrowth by reducing the oxidative stress burden, and that synaptic overgrowth is mediated by autophagy and JNK-AP-1 activity. We also examined animals defective for protection from oxidative stress and found that they too have synapse overgrowth generated by JNK-AP-1 activity. Treatment of larvae with a known ROS-generating toxin, paraquat, yielded similar synaptic responses. The observations that oxidative stress responses, potentially acting through autophagy, can generate synaptic growth suggest that ROS may be a potent regulator of synapse size and function. These findings have intriguing implications for aging neurons, neurodegenerative conditions and the interpretation of metabolic demand during learning and memory.

Supplementation of branched chain amino acids, especially leucine, is critical to improve malnutrition by regulating protein synthesis and degradation. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy. In this study, we aimed to establish a cell-free assay recapitulating leucine-mediated autophagy in vitro and dissect its biochemical requirement. We found that in a cell-free assay, membrane association of Barkor/Atg14(L), a specific autophagosome-binding protein, is suppressed by cytosol from nutrient-rich medium and such suppression is released by nutrient deprivation. We also showed that rapamycin could efficiently reverse the suppression of nutrient rich cytosol, suggesting an essential role of mTORC1 in autophagy inhibition in this cell-free system. Furthermore, we demonstrated that leucine supplementation in the cultured cells blocks Barkor puncta formation and autophagy activity. Hence, we establish a novel cell-free assay recapitulating leucine-mediated autophagy inhibition in an mTORC1-dependent manner; this assay will help us to dissect the regulation of amino acids in autophagy and related human metabolic diseases.

Gastrointestinal stromal tumor (GIST) is a prototype of mutant KIT oncogene-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Heat shock protein 90 (HSP90AA1) is a chaperone protein responsible for protein maturation and stability, and KIT is a known client protein of HSP90AA1. Inhibition of HSP90AA1 has been shown to destabilize KIT protein by enhancing its degradation via the proteasome-dependent pathway. In this study, we demonstrated that NVP-AUY922 (AUY922), a new class of HSP90AA1 inhibitor, is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the imatinib-sensitive GIST882 and imatinib-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phosphorylated KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange- or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. Therefore, the results not only highlight the potential application of AUY922 for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation.

During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.

Although hypoxia can cause cell cycle arrest, it may simultaneously suppress a conversion from this arrest to senescence. Furthermore, hypoxia can suppress senescence caused by diverse stimuli, maintaining reversible quiescence instead. Hypoxia activates autophagy and inhibits MTOR, thus also activating autophagy. What is the relationship between autophagy and cellular senescence? Also, can inhibition of MTOR and stimulation of autophagy explain the gerosuppressive effects of hypoxia?

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.

Autophagy plays an important role in the cellular response to a variety of metabolic stress conditions thus contributing to the maintenance of intracellular homeostasis. Studies in yeast have defined the genetic components involved in the initiation of autophagy as well as the progression through the autophagic cascade. The yeast kinase Atg1 initiates autophagy in response to nutrient limitation in a TOR-dependent manner. The ulk family of genes encodes the mammalian ortholog of yeast Atg1. Our recent work using mouse embryonic fibroblast (MEF) cell lines deficient for both ulk1 and ulk2 has revealed that autophagy induction is more complex in mammals than in yeast. Furthermore, these data confirm the surprising finding that a by-product of amino acid metabolism, ammonia, is a strong inducer of autophagy, as first shown by the Abraham laboratory.