bims-mascan 2019-12-08 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019‒12‒08
twenty papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

Dietary branched-chain amino acid restriction alters fuel selection and reduces triglyceride stores in hearts of Zucker fatty rats.
Reactive metabolite production is a targetable liability of glycolytic metabolism in lung cancer.
Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites.
Identification of Circulating Genomic and Metabolic Biomarkers in Intrahepatic Cholangiocarcinoma.
Validation of an analytical method using HPLC-MS/MS to quantify osimertinib in human plasma and supplementary stability results.
Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer.
Global alteration of T-lymphocyte metabolism by PD-L1 checkpoint involves a block of de novo nucleoside phosphate synthesis.
The highly unnatural fatty acid profile of cells in culture.
Metabolomics study of serum and urine samples reveals metabolic pathways and biomarkers associated with pelvic organ prolapse.
Aberrant mitochondrial function in ageing and cancer.
VOCCluster: Untargeted Metabolomics Feature Clustering Approach for Clinical Breath Gas Chromatography - Mass Spectrometry Data.
Lipidomic profiling of amniotic fluid and its application in fetal lung maturity prediction.
Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.
Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition.
Formic acid of ppm enhances LC-MS/MS detection of UV irradiation-induced DNA dimeric photoproducts.
Leveraging insights into cancer metabolism-a symposium report.
SPIN1 triggers abnormal lipid metabolism and enhances tumor growth in liver cancer.
In vitro metabolism of triclosan studied by liquid chromatography-high-resolution tandem mass spectrometry.
Editorial: Tumor Microenvironment and Resistance to Current Therapies.
A Twist of FATe: Lipid Droplets and Inflammatory Lipid Mediators.

Am J Physiol Endocrinol Metab. 2019 Dec 03.

Dietary branched-chain amino acid restriction alters fuel selection and reduces triglyceride stores in hearts of Zucker fatty rats.

Robert W McGarrah, Guo-Fang Zhang, Bridgette A Christopher, Yann Deleye, Jacquelyn M Walejko, Stephani Page, Olga R Ilkayeva, Phillip J White, Christopher B Newgard.

  Elevations in circulating levels of branched-chain amino acids (BCAA) are associated with a variety of cardiometabolic diseases and conditions. Restriction of dietary BCAA in rodent models of obesity lowers circulating BCAA levels and improves whole animal and skeletal muscle insulin sensitivity and lipid homeostasis, but the impact of BCAA supply on heart metabolism has not been studied. Here we report that feeding a BCAA-restricted chow diet to Zucker fatty rats (ZFR) causes a shift in cardiac fuel metabolism that favors fatty acid relative to glucose catabolism. This is illustrated by an increase in labeling of acetyl CoA from [1-13C]palmitate and a decrease in labeling of acetyl-CoA and malonyl-CoA from [U-13C]glucose, accompanied by a decrease in cardiac hexokinase II and GLUT4 protein levels. Metabolomic profiling of heart tissue supports these findings by demonstrating an increase in levels of a host of fatty acid-derived metabolites in hearts from ZFR and Zucker lean rats (ZLR) fed the BCAA-restricted diet. In addition, the two-fold increase in cardiac triglyceride stores in ZFR compared to ZLR fed on chow diet is eliminated in ZFR fed on the BCAA-restricted diet. Finally, the enzymatic activity of branched chain ketoacid dehydrogenase (BCKDH) is not influenced by BCAA restriction, and levels of BCAA in heart instead reflect their levels in circulation. In summary, reducing BCAA supply in obesity improves cardiac metabolic health by a mechanism independent of alterations in BCKDH activity.

Keywords:  Branched chain amino acids; Heart metabolism; Obesity; Zucker fatty rat; cardio metabolic diseases

DOI:  https://doi.org/10.1152/ajpendo.00334.2019
Nat Commun. 2019 Dec 06. 10(1): 5604

Reactive metabolite production is a targetable liability of glycolytic metabolism in lung cancer.

Alba Luengo, Keene L Abbott, Shawn M Davidson, Aaron M Hosios, Brandon Faubert, Sze Ham Chan, Elizaveta Freinkman, Lauren G Zacharias, Thomas P Mathews, Clary B Clish, Ralph J DeBerardinis, Caroline A Lewis, Matthew G Vander Heiden.

Increased glucose uptake and metabolism is a prominent phenotype of most cancers, but efforts to clinically target this metabolic alteration have been challenging. Here, we present evidence that lactoylglutathione (LGSH), a byproduct of methylglyoxal detoxification, is elevated in both human and murine non-small cell lung cancers (NSCLC). Methylglyoxal is a reactive metabolite byproduct of glycolysis that reacts non-enzymatically with nucleophiles in cells, including basic amino acids, and reduces cellular fitness. Detoxification of methylglyoxal requires reduced glutathione (GSH), which accumulates to high levels in NSCLC relative to normal lung. Ablation of the methylglyoxal detoxification enzyme glyoxalase I (Glo1) potentiates methylglyoxal sensitivity and reduces tumor growth in mice, arguing that targeting pathways involved in detoxification of reactive metabolites is an approach to exploit the consequences of increased glucose metabolism in cancer.

DOI: https://doi.org/10.1038/s41467-019-13419-4
Anal Biochem. 2019 Dec 02. pii: S0003-2697(19)30850-4. [Epub ahead of print] 113531

Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites.

Carien van der Berg, Gerda Venter, Francois H van der Westhuizen, Elardus Erasmus.

  An imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolites.

Keywords:  Biotransformation; Dansylation; Estrogen metabolism; LC-MS/MS; Method development

DOI:  https://doi.org/10.1016/j.ab.2019.113531
Cancers (Basel). 2019 Nov 28. pii: E1895. [Epub ahead of print]11(12):

Identification of Circulating Genomic and Metabolic Biomarkers in Intrahepatic Cholangiocarcinoma.

Helen Winter, Pamela J Kaisaki, Joe Harvey, Edoardo Giacopuzzi, Matteo P Ferla, Melissa M Pentony, Samantha J L Knight, Ricky A Sharma, Jenny C Taylor, James S O McCullagh.

  Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer arising from the bile ducts with a need for earlier diagnosis and a greater range of treatment options. KRAS/NRAS mutations are common in ICC tumours and 6-32% of patients also have isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations associated with metabolic changes. This feasibility study investigated sequencing circulating tumour DNA (ctDNA) combined with metabolite profiling of plasma as a method for biomarker discovery in ICC patients. Plasma was collected from four ICC patients receiving radio-embolisation and healthy controls at multiple time points. ctDNA was sequenced using Ampliseq cancer hotspot panel-v2 on Ion Torrent PGM for single nucleotide variants (SNV) detection and with Illumina whole genome sequencing for copy number variants (CNV) and further targeted examination for SNVs. Untargeted analysis of metabolites from patient and control plasma was performed using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS). Metabolite identification was performed using multi-parameter comparisons with analysis of authentic standards, and univariate statistical analysis was performed to identify differences in metabolite abundance between patient and control samples. Recurrent somatic SNVs and CNVs were identified in ctDNA from three out of four patients that included both NRAS and IDH1 mutations linked to ICC. Plasma metabolite analysis revealed biomarker metabolites associated with ICC and in particular 2-hydroxyglutarate (2-HG) levels were elevated in both samples from the only patient showing a variant allele in IDH1. A reduction in the number of CNVs was observed with treatment. This study demonstrates that ctDNA and metabolite levels can be identified and correlated in ICC patient blood samples and differentiated from healthy controls. We conclude that combining genomic and metabolic analysis of plasma offers an effective approach to biomarker identification with potential for disease stratification and early detection studies.

Keywords:  2-hydroxyglutarate; CAD; DHODH; IDH1; Intrahepatic cholangiocarcinoma; TYMS; UMPS; circulating DNA; metabolomics; orotic acid

DOI:  https://doi.org/10.3390/cancers11121895
Biomed Chromatogr. 2019 Dec 06. e4771

Validation of an analytical method using HPLC-MS/MS to quantify osimertinib in human plasma and supplementary stability results.

Ard van Veelen, Robin van Geel, Yvo de Beer, Anne-Marie Dingemans, Leo Stolk, Rob Ter Heine, Frank de Vries, Sander Croes.

  A new method for quantification of osimertinib (OSIM) in human plasma using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Methanol was used for protein precipitation and pazopanib (PAZO) as internal standard. Separation was performed on a HyPURITY® C18 analytical column (50 × 2.1 mm; 3 μm) using a gradient elution of ammonium acetate in water and ammonium acetate in methanol, both acidified with formic acid 0.1%. Detection and quantification of OSIM and PAZO was performed by a triple quadruple mass spectrometer with after electrospray ionization. This method led to robust results, as the selectivity, carry-over, precision and accuracy all met pre-specified requirements. OSIM was stable in human serum when stored at -80°C. Reduced stability was found when stored at 2-4°C or room temperature (RT). Degradation of OSIM slowed down in EDTA-plasma and acidified human serum. The limited stability of OSIM at RT should be considered for transport and sample preparation. Plasma samples should be frozen as soon as possible and sample preparation should be performed on dry-ice. In the future, EDTA-plasma and sample acidification may be used to improve OSIM stability at RT. However, more research and validation of such an approach is required.

Keywords:  analysis; osimertinib; room temperature; stability; validation

DOI:  https://doi.org/10.1002/bmc.4771
Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Nov 30. pii: S1388-1981(19)30230-6. [Epub ahead of print] 158579

Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer.

Yuting Wang, Sebastian Hinz, Ortrud Uckermann, Pia Hönscheid, Witigo von Schönfels, Greta Burmeister, Alexander Hendricks, Jacobo Miranda Ackerman, Gustavo B Baretton, Jochen Hampe, Mario Brosch, Clemens Schafmayer, Andrej Shevchenko, Sebastian Zeissig.

  Solid tumors are characterized by global metabolic alterations which contribute to their growth and progression. Altered gene expression profiles and plasma lipid composition suggested a role for metabolic reprogramming in colorectal cancer (CRC) development. However, a conclusive picture of CRC-associated lipidome alterations in the tumor tissue has not emerged. Here, we determined molar abundances of 342 species from 20 lipid classes in matched biopsies of CRC and adjacent normal mucosa. We demonstrate that in contrast to previous reports, CRC shows a largely preserved lipidome composition that resembles that of normal colonic mucosa. Important exceptions include increased levels of lyso-phosphatidylinositols in CRC and reduced abundance of ether phospholipids in advanced stages of CRC. As such, our observations challenge the concept of widespread alterations in lipid metabolism in CRC and rather suggest changes in the cellular lipid profile that are limited to selected lipids involved in signaling and the scavenging of reactive oxygen species.

Keywords:  Cancer; Colon; Lipid metabolism; Mass spectrometry; Shotgun lipidomics

DOI:  https://doi.org/10.1016/j.bbalip.2019.158579
Cell Discov. 2019 ;5 62

Global alteration of T-lymphocyte metabolism by PD-L1 checkpoint involves a block of de novo nucleoside phosphate synthesis.

Nicolaos Jay Palaskas, Jacob David Garcia, Roksana Shirazi, Daniel Sanghoon Shin, Cristina Puig-Saus, Daniel Braas, Antoni Ribas, Thomas Glen Graeber.

  Metabolic obstacles of the tumor microenvironment remain a challenge to T-cell-mediated cancer immunotherapies. To better understand the interplay of immune checkpoint signaling and immune metabolism, this study developed and used an optimized metabolite extraction protocol for non-adherent primary human T-cells, to broadly profile in vitro metabolic changes effected by PD-1 signaling by mass spectrometry-based metabolomics and isotopomer analysis. Inhibitory signaling reduced aerobic glycolysis and glutaminolysis. A general scarcity across the panel of metabolites measured supported widespread metabolic regulation by PD-1. Glucose carbon fate analysis supported tricarboxylic acid cycle reliance on pyruvate carboxylation, catabolic-state fluxes into acetyl-CoA and succinyl-CoA, and a block in de novo nucleoside phosphate synthesis that was accompanied by reduced mTORC1 signaling. Nonetheless, exogenous administration of nucleosides was not sufficient to ameliorate proliferation of T-cells in the context of multiple metabolic insufficiencies due to PD-L1 treatment. Carbon fate analysis did not support the use of primarily glucose-derived carbons to fuel fatty acid beta oxidation, in contrast to reports on T-memory cells. These findings add to our understanding of metabolic dysregulation by PD-1 signaling and inform the effort to rationally develop metabolic interventions coupled with immune-checkpoint blockade for increased treatment efficacy.

Keywords:  Cancer microenvironment; DNA metabolism; Metabolomics; RNA metabolism; Tumour immunology

DOI:  https://doi.org/10.1038/s41421-019-0130-x
Prog Lipid Res. 2019 Dec 03. pii: S0163-7827(19)30060-8. [Epub ahead of print] 101017

The highly unnatural fatty acid profile of cells in culture.

Paul L Else.

  The fatty acid profile of cells in culture are unlike any natural cell with twice the monounsaturated (MUFA) and half the polyunsaturated fatty acids (PUFA) of natural cells (Mol%). This is not due to cell lines primarily being derived from cancers but due to limited access to lipid and inability to make PUFA de novo as vertebrate cells. Classic culture methods use media with 10% serum (the only exogenous source of lipid). Fetal bovine serum (FBS), the serum of choice has a low level of lipid and cholesterol compared to other sera and at 10% of media provides 2-3% of the fatty acid and cholesterol, 1% of the PUFA and 0.03% of the essential fatty acid linoleic acid (18:2n-6) available to cells in the body. Since vertebrate cell lines cannot make PUFA they synthesise MUFA, offsetting their PUFA deficit and reducing their fatty acid diversity. Stem and primary cells in culture appear to be similarly affected, with a rapid loss of their natural fatty acid compositions. The unnatural lipid composition of cells in culture has substantial implications for examining natural stems cell in culture, and for investigations of cellular mechanisms using cell lines based on the pervasive influence of fats.

Keywords:  Cancer cell; Cell line; Fatty acid; Peroxidation; Polyunsaturated; Stem cell

DOI:  https://doi.org/10.1016/j.plipres.2019.101017
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Nov 14. pii: S1570-0232(19)31091-8. [Epub ahead of print]1136 121882

Metabolomics study of serum and urine samples reveals metabolic pathways and biomarkers associated with pelvic organ prolapse.

Wei Deng, Jun Rao, Xiaodan Chen, Dongqing Li, Ziyu Zhang, Dan Liu, Jun Liu, Yaoqing Wang, Ouping Huang.

  Pelvic organ prolapse (POP) is a common medical condition among women and involves complicated diagnostics and controversial surgical management. The exact molecular mechanism underlying POP is poorly understood, especially at the metabolism level. To explore the metabolic mechanism underlying POP and discover potential biomarkers for POP diagnosis, we applied a non-targeted metabolomics approach using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Metabolomics study of serum samples from patients with POP (n = 24) and controls (n = 22) revealed a total of 59 metabolites that are significantly different (VIP ≥ 1 and p ≤ 0.05) between the two groups. Between urine samples from POP patients (n = 45) and controls (n = 59), 33 metabolites differed significantly (VIP ≥ 1 and p ≤ 0.05). Metabolic pathways affected by these differentially expressed metabolites were analyzed. In both serum and urine samples, three pathways including arginine biosynthesis and purine metabolism were found to be significantly related to POP. Six metabolites including GPC, 1-methyladenosine, maleic acid, L-pyroglutamic acid, inosine, and citrate are significantly changed (VIP ≥ 1 and p ≤ 0.05) in both serum and urine samples from patients with POP. Receiver operating characteristics (ROC) curve analysis showed that using these six metabolites as a biomarker could distinguish patients with POP from controls with good accuracy in both serum (AUC = 1) and urine samples (AUC = 0.854). Collectively, these results further extended our understanding of key regulatory metabolic pathways involved in the pathophysiology of POP, as well as provided some promising biomarkers for effective POP diagnosis.

Keywords:  Biomarkers; POP; Serum; UHPLC-Q-TOF-MS; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2019.121882
Biogerontology. 2019 Dec 04.

Aberrant mitochondrial function in ageing and cancer.

Julia C Whitehall, Laura C Greaves.

  Alterations in mitochondrial metabolism have been described as one of the major hallmarks of both ageing cells and cancer. Age is the biggest risk factor for the development of a significant number of cancer types and this therefore raises the question of whether there is a link between age-related mitochondrial dysfunction and the advantageous changes in mitochondrial metabolism prevalent in cancer cells. A common underlying feature of both ageing and cancer cells is the presence of somatic mutations of the mitochondrial genome (mtDNA) which we postulate may drive compensatory alterations in mitochondrial metabolism that are advantageous for tumour growth. In this review, we discuss basic mitochondrial functions, mechanisms of mtDNA mutagenesis and their metabolic consequences, and review the evidence for and against a role for mtDNA mutations in cancer development.

Keywords:  Ageing; Cancer; Metabolism; Mitochondria; mtDNA mutations

DOI:  https://doi.org/10.1007/s10522-019-09853-y
Anal Chem. 2019 Dec 02.

VOCCluster: Untargeted Metabolomics Feature Clustering Approach for Clinical Breath Gas Chromatography - Mass Spectrometry Data.

Yaser Alkhalifah, Iain Phillips, Andrea Soltoggio, Kareen Darnley, William H Nailon, Duncan McLaren, Michael Eddleston, C L Paul Thomas, Dahlia Salman.

Metabolic profiling of breath analysis involves processing, alignment, scaling and clustering of thousands of features ex-tracted from Gas Chromatography Mass spectrometry (GC-MS) data from hundreds of participants. The multi-step data processing is complicated, operator error-prone and time-consuming. Automated algorithmic clustering methods that are able to cluster features in a fast and reliable way are necessary. These accelerate metabolic profiling and discovery plat-forms for next generation medical diagnostic tools. Our unsupervised clustering technique, VOCCluster, prototyped in Py-thon, handles features of deconvolved GC-MS breath data. VOCCluster was created from a heuristic ontology based on the observation of experts undertaking data processing with a suite of software packages. VOCCluster identifies and clusters groups of volatile organic compounds (VOCs) from deconvolved GC-MS breath with similar mass spectra and retention index profiles. VOCCluster was used to cluster more than 15,000 features extracted from 74 GC-MS clinical breath samples obtained from participants with cancer before and after a radiation therapy. Results were evaluated against a panel of ground truth compounds and compared to other clustering methods (DBSCAN and OPTICS) that were used in previous metabolomics studies. VOCCluster was able to cluster those features into 1081 groups (including endogenous, exogenous compounds and instrumental artefacts) with an accuracy rate of 96% (± 0.04 at 95% confidence interval).

DOI: https://doi.org/10.1021/acs.analchem.9b03084
J Clin Lab Anal. 2019 Dec 05. e23109

Lipidomic profiling of amniotic fluid and its application in fetal lung maturity prediction.

Zheng Cao, Jingrui Liu, Xin Xie, Sien Zhan, Wei Song, Shaowen Wu, Zheng Sun, Ying Dong, Guodong Tang, Yilin Liu, Lin Li, Min Shen, Yanhong Zhai, Jihua Zou, Xiaowei Liu.

  BACKGROUND: The pulmonary surfactant especially lipids in amniotic fluid can reflect the development stage of fetal lung maturity (FLM). However, the conventional lecithin/sphingomyelin (L/S) ratio method by thin layer chromatography (TLC) is insufficient and inconvenient for FLM prediction in clinical practice.METHODS: The amniotic fluid samples were collected from the pregnant women in labor or undergoing amniocentesis and analyzed for its lipid contents with the liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) method and the lamellar body count (LBC) method. To reveal the lipidomic profiling of different FLM stages, three groups of amniotic fluid samples including 8 from premature group (gestational week (GW) < 37), 10 from mature group (GW < 37), and 10 from mature group (GW > 38) were compared with the control group (n = 6) of 18 GWs separately.
RESULTS: In the FLM prediction study, the sensitivity of the LC-HRMS method and LBC method was 91% and 73%, respectively; the specificity was 100% and 95%, respectively. The most significant metabolic pathway was linoleic acid metabolism between the premature group and the control group. Both glycerophospholipid metabolism and glycosylphosphatidylinositol-anchor biosynthesis were enriched in the mature groups. In search of potential FLM prediction markers in amniotic fluid, 8 phosphatidylcholines, 1 sphingomyelin, and 1 phosphatidylethanolamine were significantly increased in the mature groups compared with the premature group.
CONCLUSION: An efficient LC-HRMS method for L/S ratio in predicting FLM was established. The linoleic acid metabolism may play an important role in the fetal lung development.

Keywords:  FLM; L/S ratio; LC-HRMS; amniotic fluid; lipidomics

DOI:  https://doi.org/10.1002/jcla.23109
Metabolites. 2019 Nov 29. pii: E293. [Epub ahead of print]9(12):

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Veronika Kulikova, Konstantin Shabalin, Kirill Nerinovski, Alexander Yakimov, Maria Svetlova, Ljudmila Solovjeva, Andrey Kropotov, Mikhail Khodorkovskiy, Marie E Migaud, Mathias Ziegler, Andrey Nikiforov.

  Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

Keywords:  NAD metabolism; NMR spectroscopy; extracellular NAD+ intermediates; human cells

DOI:  https://doi.org/10.3390/metabo9120293
Sci Adv. 2019 Nov;5(11): eaax7525

Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition.

Yi Wu, Keshi Chen, Guangsuo Xing, Linpeng Li, Bochao Ma, Zhijuan Hu, Lifan Duan, Xingguo Liu.

Metabolic reprogramming has emerged as a key regulator of cell fate decisions. Roles of glucose and amino acid metabolism have been extensively documented, whereas lipid metabolism in pluripotency remains largely unexplored. Using a high-coverage lipidomics approach, we reveal dynamic changes in phospholipids occurring during reprogramming and show that the CDP-ethanolamine (CDP-Etn) pathway for phosphatidylethanolamine (PE) synthesis is required at the early stage of reprogramming. Mechanistically, the CDP-Etn pathway inhibits NF-κB signaling and mesenchymal genes in a Pebp1-dependent manner, without affecting autophagy, resulting in accelerated mesenchymal-to-epithelial transition (MET) and enhanced reprogramming. Furthermore, PE binding to Pebp1 enhances the interaction of Pebp1 with IKKα/β and reduces the phosphorylation of IKKα/β. The CDP-Etn-Pebp1 axis is associated with EMT/MET in hepatocyte differentiation, indicating that Etn/PE is a broad-spectrum MET/EMT-regulating metabolite. Collectively, our study reveals an unforeseen connection between phospholipids, cell migration, and pluripotency and highlights the importance of phospholipids in cell fate transitions.

DOI: https://doi.org/10.1126/sciadv.aax7525
Anal Chem. 2019 Nov 30.

Formic acid of ppm enhances LC-MS/MS detection of UV irradiation-induced DNA dimeric photoproducts.

Ning Zhang, Wenchao Deng, Yao Li, Yangde Ma, Yan Liu, Xiangjun Li, Hailin Wang.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are genotoxic DNA lesions and mainly generated on thymine-thymine (T-T) dinucleotides upon UV irradiation. Regarding the sensitivity, specificity, and accuracy of analytical methods, it is of first choice to develop a reliable assay for simultaneous detection of these DNA lesions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the dilemma is the low detection sensitivity of the phosphate-containing dimeric photoproducts even using most favorable negative-ion mode for LC-MS/MS analysis. Unexpectedly, we observed that the detection sensitivity of T-T CPD and 6-4PP could be significantly improved using formic acid/acetic acid (~ppm) as an additive of the mobile phase for reversed-phase LC-MS/MS analysis. This is the first demonstration of the enhancement of LC-MS/MS signals by formic acid/acetic acid in negative-ion mode. Of note, these acidic agents are often used for positive-ion mode in LC-MS assays. Benefited from the developed method, we could quantify both T-T CPD and 6-4PP in mouse embryonic stem cells upon UVC irradiation at low dosage. This sensitive method is applicable to the screening and identification of genes involved in formation, signaling and repair of UV lesion.

DOI: https://doi.org/10.1021/acs.analchem.9b04327
Ann N Y Acad Sci. 2019 Dec 02.

Leveraging insights into cancer metabolism-a symposium report.

Jennifer Cable, Lydia Finley, Benjamin P Tu, Gary J Patti, Trudy G Oliver, Santosha Vardhana, Miyeko Mana, Russell Ericksen, Sanika Khare, Ralph DeBerardinis, Brent R Stockwell, Aimee Edinger, Marcia Haigis, William Kaelin.

  Tumor cells have devised unique metabolic strategies to garner enough nutrients to sustain continuous growth and cell division. Oncogenic mutations may alter metabolic pathways to unlock new sources of energy, and cells take the advantage of various scavenging pathways to ingest material from their environment. These changes in metabolism result in a metabolic profile that, in addition to providing the building blocks for macromolecules, can also influence cell signaling pathways to promote tumor initiation and progression. Understanding what pathways tumor cells use to synthesize the materials necessary to support metabolic growth can pave the way for new cancer therapeutics. Potential strategies include depriving tumors of the materials needed to grow or targeting pathways involved in dependencies that arise by virtue of their altered metabolis.

Keywords:  cancer; cell signaling; ferroptosis; metabolism; micropinocytosis

DOI:  https://doi.org/10.1111/nyas.14274
Cancer Lett. 2019 Nov 29. pii: S0304-3835(19)30587-7. [Epub ahead of print]

SPIN1 triggers abnormal lipid metabolism and enhances tumor growth in liver cancer.

Man Zhao, Yanan Bu, Jinyan Feng, Huihui Zhang, Yujun Chen, Guang Yang, Zixian Liu, Hongfeng Yuan, Ying Yuan, Lei Liu, Haolin Yun, Jiapei Wang, Xiaodong Zhang.

  Abnormal lipid metabolism plays crucial roles in the development of cancer. Spindlin 1 (SPIN1) involving the process of spindle organization and chromosomal stability serves as an important player in the carcinogenesis. In this study, we try to identify the new function of SPIN1 in lipid metabolism of liver cancer. Tissue microarray showed that 75% (60/80) of hepatocellular carcinoma (HCC) tissues were positive for SPIN1, which was highly expressed in clinical HCC samples and positively associated with malignancy of HCC. Strikingly, SPIN1 could modulate abnormal lipid metabolism by increasing intracellular triglycerides, cholesterols, and lipid droplets in hepatoma cells, which could remarkably enhance the proliferation of hepatoma cells. Mechanistically, SPIN1 up-regulated FASN in hepatoma cells. SPIN1 co-activated transcriptional factor SREBP1c in the promoter of FASN through interaction with SREBP1c. Moreover, SPIN1 promoted the growth of liver cancer in vitro and in vivo and the levels of intracellular triglycerides, cholesterols and lipid droplets were increased in the tumor tissues from mice. In conclusion, SPIN1 modulates abnormal lipid metabolism and enhances growth of liver cancer through SREBP1c-triggered FASN signaling. Therapeutically, SPIN1 may serve as a novel target for HCC.

Keywords:  FASN; SPIN1; SREBP1c; lipid metabolism; liver cancer

DOI:  https://doi.org/10.1016/j.canlet.2019.11.032
Anal Bioanal Chem. 2019 Dec 01.

In vitro metabolism of triclosan studied by liquid chromatography-high-resolution tandem mass spectrometry.

Amal Guesmi, Lekha Sleno.

  Triclosan (TCS) is an antibacterial and antifungal compound found in many hygiene products, including toothpaste, soap, and detergents. However, this molecule can act as an endocrine disruptor and can induce harmful effects on human health and the environment. In this study, triclosan was biotransformed in vitro using human and rat liver fractions, to evaluate oxidative metabolism, the formation of reactive metabolites via the detection of GSH adducts, as well as glucuronide and sulfate conjugates using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). A deuterated analog of triclosan was also employed for better structural elucidation of specific metabolic sites. Several GSH adducts were found, either via oxidative metabolism of triclosan or its cleavage product, 2,4-dichlorophenol. We also detected glucuronide and sulfated conjugates of triclosan and its cleaved product. This study was aimed at understanding the routes of detoxification of this xenobiotic, as well as investigating any potential pathways related to additional toxicity via reactive metabolite formation. Graphical abstract.

Keywords:  Liquid chromatography; Mass spectrometry; Metabolism; Triclosan

DOI:  https://doi.org/10.1007/s00216-019-02239-6
Front Oncol. 2019 ;9 1131

Editorial: Tumor Microenvironment and Resistance to Current Therapies.

Ahmed Lasfar, Murugabaskar Balan, Karine A Cohen-Solal, Andrew Zloza.



Keywords:  cancer cell signaling; cancer resistance; immunotherapy; targeted therapy; therapy resistance; tumor microenvironment; tumor—targeting

DOI:  https://doi.org/10.3389/fonc.2019.01131
Biochimie. 2019 Nov 28. pii: S0300-9084(19)30339-6. [Epub ahead of print]

A Twist of FATe: Lipid Droplets and Inflammatory Lipid Mediators.

Eva Jarc, Toni Petan.

  Lipid droplets are fat storage organelles present in most eukaryotic cells. They consist of a neutral lipid core containing mostly triglycerides and sterol esters and covered by a monolayer of phospholipids, wherein numerous proteins are embedded. In the cell, lipid droplets have a dynamic life cycle, rapidly altering their size, location, lipid and protein composition in response to environmental stimuli and cell state. Lipid droplets are primarily involved in the coordination of lipid metabolism with cellular requirements for energy production, membrane homeostasis and cell growth. However, they are also directly or indirectly engaged in signalling pathways. On the one hand, lipid droplets sequester lipids and proteins thereby limiting their availability for participation in signalling pathways. On the other hand, the lipolytic machinery provides a highly regulated, on-demand source of signalling lipids: lipids derived from their neutral lipid core, or the phospholipid monolayer, directly act as signalling mediators or are converted into ones. In fact, emerging studies suggest that these organelles are essential for various cellular stress response mechanisms, including inflammation and immunity, acting as hubs that integrate metabolic and inflammatory processes. Here, we discuss the ways in which lipid droplets regulate the availability of fatty acids for the activation of signalling pathways and for the production of polyunsaturated fatty acid-derived lipid mediators. We focus in particular on recent discoveries in immune cells and adipose tissue that have revealed an intricate relationship between lipid droplets and inflammatory signalling and may also be relevant for other tissues and various human diseases.

Keywords:  eicosanoids; inflammation; lipase; lipid droplets; phospholipase; resolvins

DOI:  https://doi.org/10.1016/j.biochi.2019.11.016