Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

Autophagy: A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes

The selectivity and specificity of autophagy in drosophila

Autophagy is a process of cellular self-degradation and is a major pathway for elimination of cytoplasmic material by the lysosomes. Autophagy is responsible for the degradation of damaged organelles and protein aggregates and therefore plays a significant role in cellular homeostasis. Despite the initial belief that autophagy is a nonselective bulk process, there is growing evidence during the last years that sequestration and degradation of cellular material by autophagy can be accomplished in a selective and specific manner. Given the role of autophagy and selective autophagy in several disease related processes such as tumorigenesis, neurodegeneration and infections, it is very important to dissect the molecular mechanisms of selective autophagy, in the context of the system and the organism. An excellent genetically tractable model organism to study autophagy is Drosophila, which appears to have a highly conserved autophagic machinery compared with mammals. However, the mechanisms of selective autophagy in Drosophila have been largely unexplored. The aim of this review is to summarize recent discoveries about the selectivity of autophagy in Drosophila

Tracking autophagy during proliferation and differentiation of trypanosoma brucei

Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei

Beyond self-eating: The control of nonautophagic functions and signaling pathways by autophagy-related proteins.

The identification of conserved autophagy-related proteins (ATGs) that mediate bulk degradation of cytosolic material laid the foundation for breakthroughs linking autophagy to a litany of physiological processes and disease conditions. Recent discoveries are revealing that these same ATGs orchestrate processes that are related to, and yet clearly distinct from, classic autophagy. Autophagy-related functions include secretion, trafficking of phagocytosed material, replication and egress of viral particles, and regulation of inflammatory and immune signaling cascades. Here, we define common processes dependent on ATGs, and discuss the challenges in mechanistically separating autophagy from these related pathways. Elucidating the molecular events that distinguish how individual ATGs function promises to improve our understanding of the origin of diseases ranging from autoimmunity to cancer

Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies

OBJECTIVES: Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). METHODS: For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. RESULTS: Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients. CONCLUSION: These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA

CD4 T lymphocyte autophagy is upregulated in the salivary glands of primary Sjögren’s syndrome patients and correlates with focus score and disease activity

Background: Primary Sjögren’s syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and peripheral lymphocyte perturbation. In the current study, we aimed to investigate the possible pathogenic implication of autophagy in T lymphocytes in patients with pSS. Methods: Thirty consecutive pSS patients were recruited together with 20 patients affected by sicca syndrome a nd/or chronic sialoadenitis and 30 healthy controls. Disease activity and damage were evaluated according to SS disease activity index, EULAR SS disease activity index, and SS disease damage index. T lymphocytes were analyzed for the expression of autophagy-specific markers by biochemical, molecular, and histological assays in peripheral blood and labial gland biopsies. Serum interleukin (IL)-23 and IL-21 levels were quantified by enzyme-linked immunosorbent assay. Results: Our study provides evidence for the first time that autophagy is upregulated in CD4+ T lymphocyte salivary glands from pSS patients. Furthermore, a statistically significant correlation was detected between lymphocyte autophagy levels, disease activity, and damage indexes. We also found a positive correlation between autophagy enhancement and the increased salivary gland expression of IL-21 and IL-23, providing a further link between innate and adaptive immune responses in pSS. Conclusions: These findings suggest that CD4+ T lymphocyte autophagy could play a key role in pSS pathogenesis. Additionally, our data highlight the potential exploitation of T cell autophagy as a biomarker of disease activity and provide new ground to verify the therapeutic implications of autophagy as an innovative drug target in pSS

Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy

Breast cancer patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu-overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy

An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses

Experimental Control and Characterization of Autophagy in Drosophila

Insects such as the fruit fly Drosophila melanogaster, which fundamentally reorganize their body plan during metamorphosis, make extensive use of autophagy for their normal development and physiology. In the fruit fly, the hepatic/adipose organ known as the fat body accumulates nutrient stores during the larval feeding stage. Upon entering metamorphosis, as well as in response to starvation, these nutrients are mobilized through a massive induction of autophagy, providing support to other tissues and organs during periods of nutrient deprivation. High levels of autophagy are also observed in larval tissues destined for elimination, such as the salivary glands and larval gut. Drosophila is emerging as an important system for studying the functions and regulation of autophagy in an in vivo setting. In this chapter we describe reagents and methods for monitoring autophagy in Drosophila, focusing on the larval fat body. We also describe methods for experimentally activating and inhibiting autophagy in this system and discuss the potential for genetic analysis in Drosophila to identify novel genes involved in autophagy