Steps Of Autophagy Research Articles

Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Photodynamic therapy (PDT) uses a photosensitizer, light, and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7+ cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.

Anti-Breast Cancer Activity of a Novel Strategy of Targeting Autophagy Induced by Pan-HDAC Inhibitor and Acetylated Hsp70.

Abstract During the in vivo growth of breast cancers, hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species collectively induce the metabolic and protein-misfolding/denaturing stress. This increases the levels of heat shock proteins (hsp) and induces autophagy to avoid cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote protein re-folding and prevent protein aggregation. Increase in hsp70 levels are also known to inhibit both intrinsic and extrinsic pathway of apoptosis. In our present studies, we have determined that the in vitro stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) results in hyperacetylation of hsp70, which induces autophagy of the cultured breast cancer MB-231 and MCF-7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to form the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy associated genes (ATG) 6 and ATG14, that mediates vesicle nucleation. We have determined that hyperacetylated hsp70 significantly enhances the stability and activity of VPS34, which leads to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruits the PHD domain containing E3 ligase for sumoylation, KAP1, to induce the sumoylation of VPS34 by SUMO-1. This was documented by confocal immunostaining with fluorescence-conjugated anti-VPS34 and anti-SUMO-1 antibodies, as well as by biochemical determination of in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS-induced, KAP-1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co-treatment with PS and the inhibitor of autophagy 3-methl-adenosine (3-MA) or chloroquine significantly enhanced cell death of breast cancer MB-231 and SUM159T cells. Furthermore, the in vivo role of hyperacetylated hsp70 in autophagy induced during growth of the orthotopically implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice was also established. In cohorts of mice, following growth to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates demonstrated tumor size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3 II. Collectively, these novel findings suggest that, stress-induced heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers autophagy can be selectively targeted by co-treatment with a pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3124.

Abstract B21: Targeting autophagy induced by pan-HDAC inhibitor panobinostat and promoted by acetylated hsp70: A novel therapy for breast cancer

Abstract Among the hallmarks of cancer is the stress phenotype, which is collectively induced by hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species, and exhibited as metabolic and proteinmisfolding/denaturing (proteotoxic) stress. This results in the constitutive activation of the heat shock response with elevated levels of heat shock proteins (hsp), and induction of autophagy, for avoiding cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote proper protein folding and inhibit both the intrinsic and extrinsic pathways of apoptosis. In our present studies, we have determined in vitro that the stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) resulted in hyperacetylation of hsp70, which induced autophagy in the cultured breast cancer MB-231 and MCF-7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to create the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy-associated genes (ATG) 6 and ATG14, which mediate vesicle nucleation. We determined that hyperacetylated hsp70 significantly enhanced the stability and activity of VPS34, which led to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruited the PHD domain containing E3 ligase for sumoylation, KAP1, and induced the sumoylation of VPS34 by SUMO-1. This was documented by confocal immunostaining with fluorescence-conjugated anti-VPS34 and anti-SUMO-1 antibodies, as well as by biochemical determination of the in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS-induced, KAP-1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in the cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co-treatment with PS and the inhibitor of autophagy 3- methl-adenosine (3-MA) or chloroquine significantly enhanced cell death of breast cancer MB-231 and SUM159T cells. Furthermore, we determined the in vivo role of hyperacetylated hsp70 in autophagy. In cohorts of mice, following the growth of the orthotopically-implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates were obtained. Western blot analyses demonstrated size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3II. Collectively, these findings suggest that stress phenotype associated heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers, autophagy can be selectively targeted by cotreatment with the pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B21.

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP-Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP-Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1-hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

Quantitation of autophagy by luciferase release assay

Autophagy is a cellular process that has been defined and analyzed almost entirely by qualitative measures. In no small part, this is attributable to the absence of robust quantitative assays that can easily and reliably permit the progress of key steps in autophagy to be assessed. We have recently developed a cell-based assay that specifically measures proteolytic cleavage of a tripartite sensor protein by the autophagy protease ATG4B. Activation of ATG4B results in release of Gaussia luciferase from cells that can be non-invasively harvested from cellular supernatants. Here, we compare this technique to existing methods and propose that this type of assay will be suitable for genome-wide functional screens and in vivo analysis of autophagy.

The Ras/cAMP-dependent Protein Kinase Signaling Pathway Regulates an Early Step of the Autophagy Process in Saccharomyces cerevisiae

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.

Stress-induced alterations in autophagic pathway: relationship to ubiquitin system.

The autophagic response of the cell to nutrient deprivation or heat stress is characterized by an increase in the rate of cellular protein degradation. Using temperature-sensitive mutant cell lines that harbor a mutation in the ubiquitin pathway, we have recently shown that this response is dependent on a functional ubiquitin-activating enzyme E1. The ubiquitin pathway is involved in a multitude of cellular events including protein degradation, the best understood of these. Herein the activation of the ubiquitin molecule via E1 is followed by its covalent conjugation to acceptor proteins followed by proteolysis. It is therefore important to study the linkage between the autophagic response and E1. Using these same cell lines, CHO E36 and CHO ts20, we demonstrate that after heat stress or nutrient deprivation there is a rapid and reversible decrease in the buoyant density of subcellular vesicles containing lysosomal hydrolases, a characteristic found to accompany autophagy. This stress-induced change is found in all cell lines examined, independent of the activity of the E1. The light-density vesicles, which comigrate with endosomes on colloidal silica gradients, are not accessible to the endocytic marker transferrin-horseradish peroxidase (HRP) after cellular uptake and subsequent HRP-mediated density shift analysis. Furthermore, morphology of the isolated fractions from control and stress-induced cells was similar. These results thus demonstrate the changes in hydrolase-containing intracellular vesicles that accompany nutritional deprivation or heat stress and support the notion that the linkage of the autophagic response to the ubiquitin system is at a step in autophagy which does not affect the formation of autophagic vesicles.