Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

SUMMARY Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

The strength and duration of mitogen-activated protein kinase (MAPK) signaling have been shown to regulate cell fate in different cell types. In this study, a general mechanism is described that explains how subtle differences in signaling kinetics are translated into a specific biological outcome. In fibroblasts, the expression of immediate early gene (IEG)-encoded Fos, Jun, Myc, and early growth response gene 1 (Egr-1) transcription factors is significantly extended by sustained extracellular signal-regulated kinase 1 and 2 (ERK1 and -2) signaling. Several of these proteins contain functional docking site for ERK, FXFP (DEF) domains that serve to locally concentrate the active kinase, thus showing that they can function as ERK sensors. Sustained ERK signaling regulates the posttranslational modifications of these IEG-encoded sensors, which contributes to their sustained expression during the G 1 -S transition. DEF domain-containing sensors can also interpret the small changes in ERK signal strength that arise from less than a threefold reduction in agonist concentration. As a result, downstream target gene expression and cell cycle progression are significantly changed.

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma. Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases. The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating. In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants. Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

The anti-cancer drug paclitaxel (Taxol) alters microtubule assembly and activates pro-apoptotic signaling pathways. Previously, we and others found that paclitaxel activates endogenous JNK in tumor cells, and the activation of JNK contributes to tumor cell apoptosis. Here we find that paclitaxel activates the prosurvival MEK/ERK pathway, which conversely may compromise the efficacy of paclitaxel. Hence, a combination treatment of paclitaxel and MEK inhibitors was pursued to determine whether this treatment could lead to enhanced apoptosis. The inhibition of MEK/ERK with a pharmacologic inhibitor, U0126, together with paclitaxel resulted in a dramatic enhancement of apoptosis that is four times more than the additive value of the two drugs alone. Enhanced apoptosis was verified by the terminal transferase-mediated dUTP nick end labeling assay, by an enzyme-linked immunosorbent assay for histone-associated DNA fragments, and by flow cytometric analysis for DNA content. Specificity of the pharmacologic inhibitor was confirmed by the use of (a) a second MEK/ERK inhibitor and (b) a transdominantnegative MEK. Enhanced apoptosis was verified in breast, ovarian, and lung tumor cell lines, suggesting this effect is not cell type-specific. This is the first report of enhanced apoptosis detected in the presence of paclitaxel and MEK inhibition and suggests a new anticancer strategy.Paclitaxel is a promising frontline chemotherapy in the treatment of patients with ovarian, breast, and nonsmall cell lung carcinomas (1, 2). Paclitaxel is isolated from the bark of the pacific yew (Taxus brevifolia) and functions by binding and stabilizing microtubules (3). Binding of paclitaxel to microtubules blocks normal cell cycle progression during the merger of mitotic metaphase and anaphase. This prevents chromosome segregation, leading to tumor cell death.Combination therapy of paclitaxel and Herceptin, an antiHer2-neu antibody, has produced impressive responses among breast cancer patients (4), although this combination is obviously limited to Her2-neuϩ tumors. Combination therapy with other drugs, preferably via a rational molecular basis that is widely applicable to many tumor types, is essential for improved cancer treatment. A combination of paclitaxel with reagents that activate additional apoptotic signals, or inhibit survival signals, may provide a rational molecular basis for novel chemotherapeutic strategies.A rational molecular target is the ERK 1 mitogen-activated protein (MAP) kinase pathway that may serve as an opposing force to Jun N-terminal kinase (JNK/SAPK). Previous reports have shown that JNK/SAPK leads to cell death, while MEK activation contributes to cell differentiation, proliferation, and survival (5, 6). Activated Raf-1, a serine-threonine kinase, initiates the signaling cascade through MEK, which in turn phosphorylates a second serine-threonine kinase ERK. ERK phosphorylates additional kinases and specific transcription factors, such as Elk-1 and c-Fos, which are important in cell proliferation. However, the ...

Tuberous sclerosis complex (TSC) is a genetic autosomal dominant disorder characterized by benign tumor-like lesions, called hamartomas, in multiple organ systems, including the brain, skin, heart, kidneys, and lung. These hamartomas cause a diverse set of clinical problems based on their location and often result in epilepsy, learning difficulties, and behavioral problems. TSC is caused by mutations within the TSC1 or TSC2 genes that inactivate the genes' tumor-suppressive function and drive hamartomatous cell growth. In normal cells, TSC1 and TSC2 integrate growth signals and nutrient inputs to downregulate signaling to mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine kinase that controls cell growth and cell survival. The molecular connection between TSC and mTOR led to the clinical use of allosteric mTOR inhibitors (sirolimus and everolimus) for the treatment of TSC. Everolimus is approved for subependymal giant cell astrocytomas and renal angiomyolipomas in patients with TSC. Sirolimus, though not approved for TSC, has undergone considerable investigation to treat various aspects of the disease. Everolimus and sirolimus selectively inhibit mTOR signaling with similar molecular mechanisms, but with distinct clinical profiles. This review differentiates mTOR inhibitors in TSC while describing the molecular mechanisms, pathogenic mutations, and clinical trial outcomes for managing TSC.