A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells

BACKGROUND LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia chromosome-positive cells

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease

Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins

Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFβ-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFβ. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. TRAF3 autophagy is driven by RAS and results in activation of the NF-κB family member RELB. We show that RELB represses TGFβ target promoters independently of DNA binding at NF-κB recognition sequences, instead binding with SMAD family member(s) at SMAD-response elements. Thus, autophagy antagonises TGFβ gene expression. Finally, autophagy-deficient A549 cells regain tumorigenicity upon SMAD4 knockdown. Thus, at least in this setting, a physiologic function for autophagic regulation of gene expression is tumour growth

Pig farmers’ willingness to pay for management strategies to reduce aggression between pigs

When deciding whether to invest in an improvement to animal welfare, farmers must trade-off the relative costs and benefits. Despite the existence of effective solutions to many animal welfare issues, farmers’ willingness to pay for them is largely unknown. This study modelled pig farmers’ decisions to improve animal welfare using a discrete choice experiment focused on alleviating aggression between growing/finishing pigs at regrouping. Eighty-two UK and Irish pig farm owners and managers were asked to choose between hypothetical aggression control strategies described in terms of four attributes; installation cost, on-going cost, impact on skin lesions from aggression and impact on growth rate. If they did not like any of the strategies they could opt to keep their current farm practice. Systematic variations in product attributes allowed farmers’ preferences and willingness to pay to be estimated and latent class modelling accounted for heterogeneity in responses. The overall willingness to pay to reduce lesions was low at £0.06 per pig place (installation cost) and £0.01 per pig produced (running cost) for each 1% reduction in lesions. Results revealed three independent classes of farmers. Farmers in Class 1 were unlikely to regroup unfamiliar growing/finishing pigs, and thus were unwilling to adopt measures to reduce aggression at regrouping. Farmers in Classes 2 and 3 were willing to adopt measures providing certain pre-conditions were met. Farmers in Class 2 were motivated mainly by business goals, whilst farmers in Class 3 were motivated by both business and animal welfare goals, and were willing to pay the most to reduce aggression; £0.11 per pig place and £0.03 per pig produced for each 1% reduction in lesions. Farmers should not be considered a homogeneous group regarding the adoption of animal welfare innovations. Instead, campaigns should be targeted at subgroups according to their independent preferences and willingness to pay

WNT/β-catenin signaling regulates mitochondrial activity to alter the oncogenic potential of melanoma in a PTEN-dependent manner

Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors

5-aminosalicylic acid inhibits stem cell function in human adenoma derived cells: Implications for chemoprophylaxis in colorectal tumorigenesis

Background: Most colorectal cancers (CRC) arise sporadically from precursor lesions: colonic polyps. Polyp resection prevents progression to CRC. Risk of future polyps is proportional to the number and size of polyps detected at screening, allowing identification of high-risk individuals who may benefit from effective chemoprophylaxis. We aimed to investigate the potential of 5-aminosalicylic acid (5-ASA), a medication used in the treatment of ulcerative colitis, as a possible preventative agent for sporadic CRC. Methods: Human colorectal adenoma (PC/AA/C1, S/AN/C1 and S/RG/C2), transformed adenoma PC/AA/C1/SB10 and carcinoma cell lines (LS174T and SW620) were treated with 5-ASA. The effect on growth in two- and three-dimensional (3D) culture, β-catenin transcriptional activity and on cancer stemness properties of the cells were investigated. Results: 5-ASA was shown, in vitro, to inhibit the growth of adenoma cells and suppress β-catenin transcriptional activity. Downregulation of β-catenin was found to repress expression of stem cell marker LGR5 (leucine-rich G protein-coupled receptor-5) and functionally suppress stemness in human adenoma and carcinoma cells using 3D models of tumorigenesis. Conclusions: 5-ASA can suppress the cancer stem phenotype in adenoma-derived cells. Affordable and well-tolerated, 5-ASA is an outstanding candidate as a chemoprophylactic medication to reduce the risk of colorectal polyps and CRC in those at high risk