Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy.

Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that--even after full differentiation and maturation--continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several--but not all--targets of the transcription factor FOXO1. Indeed, VEGF-deficient endothelium in vitro and in vivo showed increased levels of FOXO1 protein in the nucleus and cytoplasm. Silencing of FOXO1 in VEGF-depleted cells reversed expression profiles of several of the gene clusters that were de-regulated in VEGF knockdown, and rescued both cell death and autophagy phenotypes. Our data suggest that endothelial VEGF maintains vascular homeostasis through regulation of FOXO1 levels, thereby ensuring physiological metabolism and endothelial cell survival.

Stimulation of histone deacetylase activity by metabolites of intermediary metabolism.

Histone deacetylases (HDACs) function in a wide range of molecular processes, including gene expression, and are of significant interest as therapeutic targets. Although their native complexes, subcellular localization, and recruitment mechanisms to chromatin have been extensively studied, much less is known about whether the enzymatic activity of non-sirtuin HDACs can be regulated by natural metabolites. Here, we show that several coenzyme A (CoA) derivatives, such as acetyl-CoA, butyryl-CoA, HMG-CoA, and malonyl-CoA, as well as NADPH but not NADP(+), NADH, or NAD(+), act as allosteric activators of recombinant HDAC1 and HDAC2 in vitro following a mixed activation kinetic. In contrast, free CoA, like unconjugated butyrate, inhibits HDAC activity in vitro. Analysis of a large number of engineered HDAC1 mutants suggests that the HDAC activity can potentially be decoupled from "activatability" by the CoA derivatives. In vivo, pharmacological inhibition of glucose-6-phosphate dehydrogenase (G6PD) to decrease NADPH levels led to significant increases in global levels of histone H3 and H4 acetylation. The similarity in structures of the identified metabolites and the exquisite selectivity of NADPH over NADP(+), NADH, and NAD(+) as an HDAC activator reveal a previously unrecognized biochemical feature of the HDAC proteins with important consequences for regulation of histone acetylation as well as the development of more specific and potent HDAC inhibitors.

ZFP36-mediated mRNA decay regulates metabolism.

Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalog metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation-many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

Levocarnitine does not impair chemotherapy cytotoxicity against acute lymphoblastic leukemia.

Asparaginase (ASNase) is an integral part of pediatric induction chemotherapy that has also been shown to improve adult survival rates; however, pegylated (PEG)-ASNase induces severe hepatotoxicity in this population. Recent case reports describe the incorporation of levocarnitine (LC) supplementation into PEG-ASNase-containing induction regimens to prevent or treat hepatotoxicity. Because LC facilitates the metabolism of free fatty acids (FFA), a primary fuel source for ALL cells, LC could potentially interfere with ALL chemotherapy efficacy. To test this, we employed in vitro and in vivo models of ALL. We show in vitro that LC supplementation does not impact cytotoxicity from vincristine, daunorubicin, dexamethasone, or ASNase on human ALL cells nor lead to an increase in ALL cell metabolic rate. In vivo, we demonstrate LC does not impair PEG-ASNase monotherapy in mice with syngeneic ALL. Together, our findings show that LC supplementation is a safe strategy to prevent/reverse ASNase-induced toxicities in preclinical models.

Lactate dehydrogenase activity drives hair follicle stem cell activation.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation.

Rethinking glutamine addiction.

Tumours reprogram their metabolism to maximize macromolecule biosynthesis for growth. However, which of the common tumour-associated metabolic activities are critical for proliferation remains unclear. Glutamate-derived glutamine is now shown to satisfy the glutamine needs of glioblastoma, indicating that glutamine anaplerosis is dispensable for growth.