ABSTRACT
One-carbon (1C) metabolism is a network of biochemical reactions distributed across organelles that delivers folate-activated 1C units to support macromolecule synthesis, methylation, and reductive homeostasis. Fluxes through these pathways are up-regulated in highly proliferative cancer cells, and anti-folates, which target enzymes within the 1C pathway, have long been used in the treatment of cancer. In this work, we review fundamental aspects of 1C metabolism and place it in context with other biosynthetic and redox pathways, such that 1C metabolism acts to bridge pathways across compartments. We further discuss the importance of stable-isotope-tracing techniques combined with mass spectrometry analysis to study 1C metabolism and conclude by highlighting therapeutic approaches that could exploit cancer cells' dependency on 1C metabolism.
ABSTRACT
AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.
Subject(s)
Cardiomyopathy, Dilated , Molecular Targeted Therapy , Myocytes, Cardiac , Protein Kinase Inhibitors , Serine , Troponin T , Activating Transcription Factor 4/metabolism , Adenosine Triphosphate/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Carbazoles/pharmacology , Carbazoles/therapeutic use , Cardiomyopathy, Dilated/drug therapy , Cardiomyopathy, Dilated/genetics , Drug Evaluation, Preclinical/methods , Glucose/metabolism , Glycine/biosynthesis , Glycine/genetics , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Induced Pluripotent Stem Cells/physiology , Mutation , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Phosphoglycerate Dehydrogenase/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Serine/antagonists & inhibitors , Serine/biosynthesis , Serine/genetics , Troponin T/genetics , Troponin T/metabolismABSTRACT
Mitochondria are central to metabolic homeostasis, and progressive mitochondrial defects have diverse metabolic consequences that could drive distinct pathophysiological states. Here, we comprehensively characterized metabolic alterations in PolgD257A mice. Plasma alanine increased markedly with time, with other organic acids accumulating to a lesser extent. These changes were reflective of increased Cori and Cahill cycling in PolgD257A mice and subsequent hypoglycemia, which did not occur during normal mouse aging. Tracing with [15N]ammonium further supported this shift in amino acid metabolism with mild impairment of the urea cycle. We also measured alterations in the lipidome, observing a reduction in canonical lipids and accumulation of 1-deoxysphingolipids, which are synthesized from alanine via promiscuous serine palmitoyltransferase activity and correlate with peripheral neuropathy. Consistent with this metabolic link, PolgD257A mice exhibited thermal hypoalgesia. These results highlight the longitudinal changes that occur in intermediary metabolism upon mitochondrial impairment and identify a contributing mechanism to mitochondria-associated neuropathy.
ABSTRACT
Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.
Subject(s)
Haploinsufficiency , Phosphoglycerate Dehydrogenase/genetics , Retinal Telangiectasis/genetics , Serine/biosynthesis , Cohort Studies , Humans , Phenotype , Retinal Pigment Epithelium/metabolismABSTRACT
OBJECTIVE: Skeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. METHODS: Primary human MPCs (hMPCs) were used for in vitro experiments, and young (4-6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. RESULTS: We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. CONCLUSIONS: These studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration.
Subject(s)
Muscle Development/physiology , Muscle, Skeletal/metabolism , Stem Cells/metabolism , Adult , Aged , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Female , Glycine/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/physiology , Myoblasts/cytology , Primary Cell Culture , Regeneration/physiology , Serine/metabolism , Stem Cells/pathologyABSTRACT
The single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer's. Despite its biological importance, there is a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in cells from patients with Niemann-Pick type C1 (NPC1), a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.
Subject(s)
Aldehydes/chemistry , Fluorescent Dyes/chemistry , Small Molecule Libraries/chemistry , Sphingosine/analysis , HeLa Cells , Humans , Microscopy, Fluorescence , Molecular Structure , Optical ImagingABSTRACT
Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.
Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Serine/deficiency , Sphingolipids/chemistry , Sphingolipids/metabolism , Alanine/biosynthesis , Alanine/metabolism , Alanine/pharmacology , Animals , Cell Adhesion/drug effects , Cell Division/drug effects , Diet , Female , Glycine/biosynthesis , Glycine/deficiency , Glycine/metabolism , Glycine/pharmacology , HCT116 Cells , Humans , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Mice , Mitochondria/metabolism , Neoplasms/drug therapy , Phosphoglycerate Dehydrogenase/antagonists & inhibitors , Phosphoglycerate Dehydrogenase/metabolism , Pyruvic Acid/metabolism , Serine/blood , Serine/pharmacology , Serine C-Palmitoyltransferase/antagonists & inhibitors , Serine C-Palmitoyltransferase/metabolism , Spheroids, Cellular/pathology , Sphingolipids/biosynthesis , Stress, Physiological/drug effects , Xenograft Model Antitumor AssaysABSTRACT
SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.
Subject(s)
Amino Acid Transport System y+/genetics , Carcinoma, Renal Cell/genetics , Cystine/metabolism , Gene Expression Regulation, Neoplastic , Kidney Neoplasms/genetics , Pentose Phosphate Pathway/genetics , Amino Acid Transport System y+/antagonists & inhibitors , Amino Acid Transport System y+/metabolism , Animals , Biological Transport/drug effects , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/mortality , Carcinoma, Renal Cell/secondary , Cell Death/drug effects , Cell Line, Tumor , Disulfides/metabolism , Gastrointestinal Agents/pharmacology , Glucose/deficiency , Glucose Transporter Type 1/antagonists & inhibitors , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 3/antagonists & inhibitors , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Humans , Kidney Neoplasms/metabolism , Kidney Neoplasms/mortality , Kidney Neoplasms/pathology , Mice , Mice, Nude , Phosphogluconate Dehydrogenase/genetics , Phosphogluconate Dehydrogenase/metabolism , Pyrazoles/pharmacology , Quinolines/pharmacology , Stress, Physiological , Sulfasalazine/pharmacology , Survival Analysis , Xenograft Model Antitumor AssaysABSTRACT
Oxidation-reduction (redox) reactions are ubiquitous in biology and typically occur in specific subcellular compartments. In cells, the electron transfer between molecules and organelles is commonly facilitated by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). While often taken for granted, these metabolic reactions are critically important for maintaining redox homeostasis and biochemical potentials across membranes. While 13C tracing and metabolic flux analysis (MFA) have emerged as powerful tools to study intracellular metabolism, this approach is limited when applied to pathways catalyzed in multiple cellular compartments. To address this issue, we and others have applied 2H (deuterium) tracers to observe transfer of labeled hydride anions, which accompanies electron transfer. Furthermore, we have developed a reporter system for indirectly quantifying NADPH enrichment in specific subcellular compartments. Here, we provide a detailed description of 2H tracing applications and the interrogation of mitochondrial versus cytosolic NAD(P)H metabolism in cultured mammalian cells. Specifically, we describe the generation of reporter cell lines that express epitope-tagged R132H-IDH1 or R172K-IDH2 and produce (D)2-hydroxyglutarate in a doxycycline-dependent manner. These tools and methods allow for quantitation of reducing equivalent turnover rates, the directionality of pathways present in multiple compartments, and the estimation of pathway contributions to NADPH pools.
Subject(s)
Deuterium/metabolism , Mammals/metabolism , NADP/metabolism , Animals , Cell Line , Cytosol/metabolism , Electron Transport/physiology , HEK293 Cells , Humans , Mitochondria/metabolism , NAD/metabolism , Oxidation-ReductionABSTRACT
The collagen-rich tumor microenvironment plays a critical role in directing the migration behavior of cancer cells. 3D collagen architectures with small pores have been shown to confine cells and induce aggressive collective migration, irrespective of matrix stiffness and density. However, it remains unclear how cells sense collagen architecture and transduce this information to initiate collective migration. Here, we tune collagen architecture and analyze its effect on four core cell-ECM interactions: cytoskeletal polymerization, adhesion, contractility, and matrix degradation. From this comprehensive analysis, we deduce that matrix architecture initially modulates cancer cell adhesion strength, and that this results from architecture-induced changes to matrix degradability. That is, architectures with smaller pores are less degradable, and degradability is required for cancer cell adhesion to 3D fibrilar collagen. The biochemical consequences of this 3D low-attachment state are similar to those induced by suspension culture, including metabolic and oxidative stress. One distinction from suspension culture is the induction of collagen catabolism that occurs in 3D low-attachment conditions. Cells also upregulate Snail1 and Notch signaling in response to 3D low-attachment, which suggests a mechanism for the emergence of collective behaviors.