Rapamycin inhibits mTORC1, but not completely.

Rapamycin is widely used as a complete inhibitor of the mTORC1 nutrient-sensitive signaling complex. Using a novel ATP-competitive inhibitor named Torin1, we have found that many mTORC1 functions that regulate cap-dependent translation and autophagy are resistant to inhibition by rapamycin.

The pharmacology of mTOR inhibition.

A flurry of reports indicates that we are entering a new phase in the development of mammalian target of rapamycin (mTOR)-based therapies for oncology. Here, we summarize exciting findings regarding mTOR signaling and the outlook for mTOR inhibitors as tools to study the mTOR pathway and as drugs in the clinic.

Tumours with PI3K activation are resistant to dietary restriction.

Dietary restriction delays the incidence and decreases the growth of various types of tumours, but the mechanisms underlying the sensitivity of tumours to food restriction remain unknown. Here we show that certain human cancer cell lines, when grown as tumour xenografts in mice, are highly sensitive to the anti-growth effects of dietary restriction, whereas others are resistant. Cancer cells that form dietary-restriction-resistant tumours carry mutations that cause constitutive activation of the phosphatidylinositol-3-kinase (PI3K) pathway and in culture proliferate in the absence of insulin or insulin-like growth factor 1.

Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning.

Many biological pathways were first uncovered by identifying mutants with visible phenotypes and by scoring every sample in a screen via tedious and subjective visual inspection. Now, automated image analysis can effectively score many phenotypes. In practical application, customizing an image-analysis algorithm or finding a sufficient number of example cells to train a machine learning algorithm can be infeasible, particularly when positive control samples are not available and the phenotype of interest is rare.

mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice.

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice.

An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1.

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin.

Rag proteins regulate amino-acid-induced mTORC1 signalling.

The serum- and nutrient-sensitive protein kinase mTOR (mammalian target of rapamycin) is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of the Rag (Ras-related GTPase) family of GTPases as mediators of amino acid signalling to mTOR is an important step towards our understanding of this mechanism.

Highly parallel identification of essential genes in cancer cells.

More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells.

CellProfiler Analyst: data exploration and analysis software for complex image-based screens.

Background: Image-based screens can produce hundreds of measured features for each of hundreds of millions of individual cells in a single experiment.

Results: Here, we describe CellProfiler Analyst, open-source software for the interactive exploration and analysis of multidimensional data, particularly data from high-throughput, image-based experiments.

Conclusion: The system enables interactive data exploration for image-based screens and automated scoring of complex phenotypes that require combinations of multiple measured features per cell.

Cancer cell metabolism: Warburg and beyond.

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins--a family of four related small guanosine triphosphatases (GTPases)--interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation.

Plakoglobin is required for effective intermediate filament anchorage to desmosomes.

Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes.

Role of the air-water interface in the retention of TiO2 nanoparticles in porous media during primary drainage.

The increasing use of nanomaterials in commercial products has resulted in increased concerns about their potential environmental impacts. The overall mobility of nanomaterials in the environment may depend in part on their mobility in the unsaturated zone of the subsurface, which may provide a sink for nanomaterials, preventing their spread, or a long-term contaminant source. The objective of this work was to study the dynamic unsaturated transport of titanium dioxide (TiO2) during primary drainage to examine the role of air-water interface formation on nanomaterial retention.

Increased mTORC1 signaling UPRegulates stress.

In this issue of Molecular Cell, Ozcan et al. (2008) show that the loss of the tuberous sclerosis tumor suppressor complex induces endoplasmic reticulum stress, leading to attenuation of insulin receptor signaling activity via the unfolded protein response.

Rhamnolipid biosurfactant mixtures for environmental remediation.

This study investigated the efficiency of rhamnolipid biosurfactant and synthetic surfactant mixtures for improving the interfacial activity of the surfactant system against several light non-aqueous-phase liquids (LNAPLs). Since the rhamnolipid biosurfactant proved to be relatively hydrophilic, we hypothesized that mixtures of rhamnolipid biosurfactants with more hydrophobic synthetic surfactants would produce lower interfacial tensions (IFTs) than an individual rhamnolipid biosurfactant. The minimum IFT observed for rhamnolipid alone and toluene (0.

Adsorption of beta blockers to environmental surfaces.

Beta-adrenergic blocking agents (beta blockers) are widely used pharmaceuticals which have been detected in the environment. Predicting the transport and ultimate fate of beta blockers in the environment requires understanding their adsorption to soils and sediments, something for which little information is currently available. The objective of this work was to examine the adsorption of three beta blockers, propranolol, metoprolol and nadolol, to a natural alluvial material, as well as to six minerals present as components of the alluvial material.

Defining the role of mTOR in cancer.

The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cell-signaling pathways commonly deregulated in human cancers. This has led to the prediction that mTOR inhibitors may be useful in oncology, and derivatives of one such molecule, rapamycin (from which mTOR derives its name), are currently in clinical development. In this review, we discuss recent progress in understanding mTOR signaling, paying particular attention to its relevance in cancer.

Basis for formulating biosurfactant mixtures to achieve ultra low interfacial tension values against hydrocarbons.

Biosurfactants could potentially replace or be used in conjunction with synthetic surfactants to provide for more cost-effective subsurface remediation. The design of surfactant formulations that are effective in lowering interfacial tension (IFT), which is necessary to mobilize entrapped hydrocarbons, requires information about the surface-active agent (surfactant) and the targeted non-aqueous phase liquids (NAPL). We hypothesized that biosurfactant and synthetic surfactant mixtures can be formulated to provide the appropriate hydrophobic/hydrophilic conditions necessary to produce low IFT against NAPLs, and that such mixtures will produce synergism that make them more effective than individual biosurfactants or synthetic surfactants.

Sorption and transport of acetaminophen, 17alpha-ethynyl estradiol, nalidixic acid with low organic content aquifer sand.

The sorption and transport of three pharmaceutical compounds (acetaminophen, an analgesic; nalidixic acid, an antibiotic; and 17alpha-ethynyl estradiol, a synthetic hormone) were examined by batch sorption experiments and solute displacement in columns of silica, alumina, and low organic carbon aquifer sand at neutral pH. Silica and alumina were used to represent negatively-charged and positively-charged fractions of subsurface media. Column transport experiments were also conducted at pH values of 4.

PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations.

Off-target effects associated with long dsRNAs in Drosophila RNAi screens.

Evidence of off-target effects (OTEs) associated with small interfering (si)RNAs (19-29bp) in mammalian cells has existed for several years. Two recent articles demonstrate that short sequences within long double-stranded (ds)RNAs frequently cause undesirable OTEs in cultured Drosophila cells. These results reveal the potential for high false-positive rates in RNA interference (RNAi) screens using long dsRNAs and highlight the need for screening with multiple, non-overlapping long dsRNAs or siRNAs.

CellProfiler: free, versatile software for automated biological image analysis.

Careful visual examination of biological samples is quite powerful, but many visual analysis tasks done in the laboratory are repetitive, tedious, and subjective. Here we describe the use of the open-source software, CellProfiler, to automatically identify and measure a variety of biological objects in images. The applications demonstrated here include yeast colony counting and classifying, cell microarray annotation, yeast patch assays, mouse tumor quantification, wound healing assays, and tissue topology measurement.

Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML.

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation.

Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, we generated mice deficient for raptor, rictor, or mLST8.