Andrew N. Lane

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at http://msi-workgroups.sourceforge.net/ or http://Msi-workgroups-feedback@lists.sourceforge.net. Further, community input related to this document can also be provided via this electronic forum.

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

Stability and kinetics of G-quadruplex structures

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM)

BackgroundMetabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma.ResultsMany primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein.ConclusionPC activation – revealed here for the first time in human subjects – may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth

6-Phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD+, and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 μmol/L) and is selectively cytostatic to ras-transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents. [Mol Cancer Ther 2008;7(1):110–20]

Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC.

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ1-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using 13C,15N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies.

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

Targeting aspartate aminotransferase in breast cancer

IntroductionGlycolysis is increased in breast adenocarcinoma cells relative to adjacent normal cells in order to produce the ATP and anabolic precursors required for survival, growth and invasion. Glycolysis also serves as a key source of the reduced form of cytoplasmic nicotinamide adenine dinucleotide (NADH) necessary for the shuttling of electrons into mitochondria for electron transport. Lactate dehydrogenase (LDH) regulates glycolytic flux by converting pyruvate to lactate and has been found to be highly expressed in breast tumours. Aspartate aminotransferase (AAT) functions in tandem with malate dehydrogenase to transfer electrons from NADH across the inner mitochondrial membrane. Oxamate is an inhibitor of both LDH and AAT, and we hypothesised that oxamate may disrupt the metabolism and growth of breast adenocarcinoma cells.MethodsWe examined the effects of oxamate and the AAT inhibitor amino oxyacetate (AOA) on 13C-glucose utilisation, oxygen consumption, NADH and ATP in MDA-MB-231 cells. We then determined the effects of oxamate and AOA on normal human mammary epithelial cells and MDA-MB-231 breast adenocarcinoma cell proliferation, and on the growth of MDA-MB-231 cells as tumours in athymic BALB/c female mice. We ectopically expressed AAT in MDA-MB-231 cells and examined the consequences on the cytostatic effects of oxamate. Finally, we examined the effect of AAT-specific siRNA transfection on MDA-MB-231 cell proliferation.ResultsWe found that oxamate did not attenuate cellular lactate production as predicted by its LDH inhibitory activity, but did have an anti-metabolic effect that was similar to AAT inhibition with AOA. Specifically, we found that oxamate and AOA decreased the flux of 13C-glucose-derived carbons into glutamate and uridine, both products of the mitochondrial tricarboxylic acid cycle, as well as oxygen consumption, a measure of electron transport chain activity. Oxamate and AOA also selectively suppressed the proliferation of MDA-MB-231 cells relative to normal human mammary epithelial cells and decreased the growth of MDA-MB-231 breast tumours in athymic mice. Importantly, we found that ectopic expression of AAT in MDA-MB-231 cells conferred resistance to the anti-proliferative effects of oxamate and that siRNA silencing of AAT decreased MDA-MB-231 cell proliferation.ConclusionsWe conclude that AAT may be a valid molecular target for the development of anti-neoplastic agents.

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Under normoxia, 2-deoxy-d-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation

In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response–specific mediator of apoptosis, more effectively in 2-DG–sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor. [Mol Cancer Ther 2007;6(11):3049–58]