Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.Metabolism in cancer cells differs from that of normal nonproliferative cells. Perhaps the most common variation from the norm in cancer metabolism is "aerobic glycolysis" or the Warburg effect. Under the Warburg effect, metabolism of glucose is largely fermentative rather than respiratory, with increased production of lactate, in normal atmospheric oxygen conditions (1). This is also associated with increased uptake of glucose, a common characteristic of cancers detectable in tumors in patients via 18 F-deoxyglucose-PET (2). However, the extent to which the Warburg effect represents a rebalancing of metabolism (increasing fermentation while decreasing respiration) versus an amplification of metabolism (increasing fermentation while maintaining, or even increasing, respiration) is the subject of debate (3, 4). The Warburg effect contrasts with the Pasteur effect, in that the latter describes the switch from fermentation to respiration when oxygen is plentiful, and its reversal when oxygen is limiting (5), while the Warburg effect describes fermentative activity of cancer cells irrespective of oxygen. In the progression of tumors, cancer cells are subject to a range of oxygen concentrations, and low oxygen induces hypoxia-inducible factor (HIF), 2 which leads to a metabolic rewiring of cancer cells, resulting in a more glycolytic metabolism (6). Therefore, cancer cells may potentially demonstrate both Warburg and Pasteur effects. Furthermore, beyond glycolysis, altered oncogene expression has strong effects on other branches of central carbon metabolism. For insta...

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxiainducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-b-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3b. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.

Mutations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of diverse ventral midline structures. The MID1 protein has been shown to function as an E3 ligase targeting the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitinmediated degradation. However, the molecular pathways downstream of the MID1/PP2A axis that are dysregulated in OS and that translate dysfunctional MID1 and elevated levels of PP2A-C into the OS phenotype are poorly understood. Here, we show that perturbations in MID1/PP2A affect mTORC1 signaling. Increased PP2A levels, resulting from proteasome inhibition or depletion of MID1, lead to disruption of the mTOR/Raptor complex and downregulated mTORC1 signaling. Congruously, cells derived from OS patients that carry MID1 mutations exhibit decreased mTORC1 formation, S6K1 phosphorylation, cell size, and cap-dependent translation, all of which is rescued by expression of wild-type MID1 or an activated mTOR allele. Our findings define mTORC1 signaling as a downstream pathway regulated by the MID1/PP2A axis, suggesting that mTORC1 plays a key role in OS pathogenesis.ubiquitin ligase | proteasome-mediated degradation M utations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of ventral midline structures. The clinical manifestations of Opitz syndrome are characterized by a diverse spectrum of symptoms, including hypertelorism and hypospadias, cleft lip/palate, dysphagia, heart defects, and mental retardation (1).The MID1 protein is a member of the RBCC/TRIM family, which contains a conserved module of the RING domain, followed by B-boxes and a coiled-coil domain (2). MID1 also contains fibronectin type III and B30.2/SPRY domains. The RING domain has been well characterized in ubiquitin-mediated protein degradation, whereas the other domains mediate proteinprotein interactions (3-6). Opitz syndrome-derived mutations in MID1 have been identified throughout the protein and show several functional consequences such as compromised association with microtubules and/or transport along microtubules (4, 7-9). In addition to its microtubule-binding function, MID1 also functions as an E3 ligase that targets the microtubule-associated pool of the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation through an interaction with the protein α4 (3). Perturbation of the E3 ligase function of MID1 in OS cells leads to the accumulation of PP2A-C and the dramatic dephosphorylation of microtubule-associated proteins, which is postulated to contribute to OS pathogenesis (3).Signaling from the mammalian target of rapamycin (mTOR) controls diverse cellular processes such as growth, autophagy, stress responses, cytoskeletal reorganization, cell motility, metabolism, and aging (10-12). mTORC1 consists of mTOR and the interacting proteins, Raptor, and mLST8. In particular, Raptor plays an essential scaffolding...

A new series of 3-ethynyl-1H–indazoles has been synthesized and evaluated in both biochemical and cell-based assays as potential kinase inhibitors. Interestingly, a selected group of compounds identified from this series exhibited low micromolar inhibition against critical components of the PI3K pathway, targeting PI3K, PDK1 and mTOR kinases. Combination of computational modeling and structure-activity relationships studies reveal a possible novel mode for PI3K inhibition, resulting in a PI3Kα isoform specific compound. Hence, by targeting the most oncogenic mutant isoform of PI3K, the compound displays anti-proliferative activity both in monolayer human cancer cell cultures and in three-dimensional tumor models. Because of its favorable physicochemical, in vitro ADME and drug-like properties, we propose that this novel ATP mimetic scaffold could result useful in deriving novel selecting and multi-kinase inhibitors for clinical use.