The Pro-apoptotic STK38 Kinase Is a New Beclin1 Partner Positively Regulating Autophagy.

Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated.

Unsaturated fatty acids induce non-canonical autophagy.

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus.

Autophagy and autophagic flux in tumor cells.

Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo.

BAG6/BAT3 modulates autophagy by affecting EP300/p300 intracellular localization.

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression.

BAT3 modulates p300-dependent acetylation of p53 and autophagy-related protein 7 (ATG7) during autophagy.

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7.

Regulation of autophagy by cytosolic acetyl-coenzyme A.

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.

Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy.

Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.

Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance.

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs.

Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

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.

PP2A blockade inhibits autophagy and causes intraneuronal accumulation of ubiquitinated proteins.

Using cultured cortical neurons, we show that the blockade of protein phosphatase 2A (PP2A), either pharmacologically by okadaic acid or by short hairpin RNA (shRNA)-mediated silencing of PP2A catalytic subunit, inhibited basal autophagy and autophagy induced in several experimental settings (including serum deprivation, endoplasmic reticulum stress, rapamycin, and proteasome inhibition) at early stages before autophagosome maturation. Conversely, PP2A upregulation by PP2A catalytic subunit overexpression stimulates neuronal autophagy. In addition, PP2A blockade resulted in the activation of the negative regulator of autophagy mammalian target of rapamycin complex 1 and 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) and led to intraneuronal accumulation of p62- and ubiquitin-positive protein inclusions, likely due to autophagy downregulation.

Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells.

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.

The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells.

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells.

Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase.

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.

Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers.

Ceramide-induced autophagy: to junk or to protect cells?

Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased.

Assaying of autophagic protein degradation.

Macroautophagy is a three-step process: (1) autophagosomes form and mature, (2) the autophagosomes fuse with lysosomes, and (3) the autophagic cargo is degraded in the lysosomes. It is this lysosomal degradation of the autophagic cargo that constitutes the autophagic flux. As in the case of metabolic pathways, the steady-state concentration of the intermediary autophagic structures alone is insufficient for investigating the flux.

Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy.

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C(2)-ceramide and C(6)-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2.

Regulation of autophagy by NFkappaB transcription factor and reactives oxygen species.

The NF-kappaB transcription factor is an important anti-apoptotic factor, which is frequently deregulated in cancer cells. We have recently demonstrated that NF-kappaB activation mediates the repression of autophagy in response to TNFa in three models of cancer cell lines. In contrast, in the absence of NF-kappaB activation, TNFa induces macroautophagy (autophagy), which requires reactive oxygen species (ROS) production and participates in the TNFalpha-induced apoptotic signaling pathway.

AMP-activated protein kinase and the regulation of autophagic proteolysis.

Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK.

NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy.

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR.

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

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).

Activity and tissue distribution of splice variants of alpha6-fucosyltransferase in human embryogenesis.

The product of the FUT8 gene transfers an alpha1-6 fucose on the innermost N-acetylglucosamine of the chitobiose core of N-glycans. Northern blot analysis shows four main transcripts of 3.0, 3.

The G-protein regulator AGS3 controls an early event during macroautophagy in human intestinal HT-29 cells.

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the alpha-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation.

Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells.

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Galpha-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J.

Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line.

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity.