A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.Autophagy refers to an evolutionarily conserved process in which intracellular proteins and organelles are sequestered in autophagosomes and subsequently degraded by lysosomal enzymes for the purpose of recycling cellular components to sustain metabolism during nutrient deprivation and to prevent accumulation of damaged proteins and organelles (1, 2). Autophagy is a dynamic process, consisting of several sequential stages (initiation, nucleation, elongation, and maturation) controlled by a group of autophagy-related genes (Atg genes). So far, more than 30 Atg genes have been identified in yeast, and many of them have homologues in mammalian cells (3). Upstream of ATG proteins, mammalian target of rapamycin (mTOR) 4 has been well studied as the key regulatory molecule (4). mTOR is a serine/threonine protein kinase serving as the convergence point for many of the upstream stimuli and pathways to regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis, translation, and autophagy (5-7). Abundance of nutrients, including growth factors, glucose, and amino acids will activate mTOR and suppress autophagy, whereas nutrient deprivation will suppress mTOR, leading to activation of autophagy. At present, the molecular mechanisms downstream of mTOR responsible for its anti-autophagic function have not been fully understood. In yeast, TOR directly targets the ATG13-ATG1 complex and suppresses its function at the initiation stage of autophagy (8). In mammalian cells, the complex containing ULK1 (the ATG1 homologue), ATG13, and FIP200 is directly cont...

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ( Myr PKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C 2 -ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B 1 . Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C 2 -ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C 2 -ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifendependent up-regulation. Tamoxifen and C 2 -ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles (1-3). Autophagy starts with the formation of a multilayer membrane-bound autophagosome that sequesters fractions of the cytoplasm (4, 5). In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed (6, 7).The physiological importance of autophagy during starvation has been primarily highlighted in rat liver (8) and then in different cell types (reviewed in Refs. 9 and 10). At the same time, the term autophagic cell death or type II programmed cell death (PCD II) has been introduced (11) to describe a cell death different from apoptosis or type I programmed cell death (PCD I) (reviewed in Refs. 12 and 13). The recent progress made in characterization of the molecular mechanism controlling autophagy has brought a renewal of interest for this process (14). There is now evidence for the role of autophagy during development (15-17), in the life span extension (15,18), and in disease such as cancer (19,20), neurodegenerative disease (21, 22), and myopathies (23,24).A family of autophagy-related genes discovered in yeast and almost integrally conserved in all eucaryotic phyla controls the formation of the autophagosome (25). Two conjugation systems (Atg5p-Atg12p and Atg8p lipidation) are involved in...

These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-B activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-B.

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Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed ‘mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1+) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.