bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒12‒01
twenty-nine papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

  1. Cell Metab. 2019 Nov 12. pii: S1550-4131(19)30568-6. [Epub ahead of print]
      The remarkable cellular and genetic heterogeneity of soft tissue sarcomas (STSs) limits the clinical benefit of targeted therapies. Here, we show that expression of the gluconeogenic isozyme fructose-1,6-bisphosphatase 2 (FBP2) is silenced in a broad spectrum of sarcoma subtypes, revealing an apparent common metabolic feature shared by diverse STSs. Enforced FBP2 expression inhibits sarcoma cell and tumor growth through two distinct mechanisms. First, cytosolic FBP2 antagonizes elevated glycolysis associated with the "Warburg effect," thereby inhibiting sarcoma cell proliferation. Second, nuclear-localized FBP2 restrains mitochondrial biogenesis and respiration in a catalytic-activity-independent manner by inhibiting the expression of nuclear respiratory factor and mitochondrial transcription factor A (TFAM). Specifically, nuclear FBP2 colocalizes with the c-Myc transcription factor at the TFAM locus and represses c-Myc-dependent TFAM expression. This unique dual function of FBP2 provides a rationale for its selective suppression in STSs, identifying a potential metabolic vulnerability of this malignancy and possible therapeutic target.
    Keywords:  Myc; TFAM; fructose-1,6-bisphosphatase 2; glycolysis; mitochondrial function; sarcoma progression
  2. J Biol Chem. 2019 Nov 24. pii: jbc.RA119.010892. [Epub ahead of print]
      mTOR Complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn, activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of TSC complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.
    Keywords:  Phospholipase D; amino acid; cancer biology; cancer therapy; growth factor; lysosome; mTOR complex (mTORC); phosphatidic acid; phospholipid vesicle
  3. Angew Chem Int Ed Engl. 2019 Nov 25.
      Metabolomics is a powerful systems biology approach that monitors changes in biomolecule concentrations to diagnose and monitor health and disease. The leading metabolomics technologies, such as NMR and mass spectrometry (MS), currently access only a small portion of the metabolome, suggesting that new technologies with orthogonal and chemically specific capabilities can provide increased metabolome coverage and further advance the diagnostic power of metabolomics. Here we report a novel approach using the high sensitivity and chemical specificity of surface enhanced Raman scattering (SERS) for online detection of metabolites from tumor lysates following liquid chromatography (LC). Our results demonstrate that this LC-SERS approach has metabolite detection capabilities comparable to the state-of-art LC-MS but suggest a selectivity for the detection of a different subset of metabolites. Analysis of replicate LC-SERS experiments exhibit reproducible metabolite patterns that we convert into barcodes, which can differentiate different tumor models. Our work demonstrates the potential of LC-SERS technology for metabolomics-based diagnosis and treatment of cancer.
    Keywords:  Chromatography; Metabolism; Raman spectroscopy; Surface-Enhanced Raman Scattering; sensors
  4. Metabolites. 2019 Nov 27. pii: E291. [Epub ahead of print]9(12):
      The onset of ulcerative colitis (UC) is characterized by a dysregulated mucosal immune response triggered by several genetic and environmental factors in the context of host-microbe interaction. This complexity makes UC ideal for metabolomic studies to unravel the disease pathobiology and to improve the patient stratification strategies. This study aims to explore the mucosal metabolomic profile in UC patients, and to define the UC metabolic signature. Treatment- naïve UC patients (n = 18), UC patients in deep remission (n = 10), and healthy volunteers (n = 14) were recruited. Mucosa biopsies were collected during colonoscopies. Metabolomic analysis was performed by combined gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). In total, 177 metabolites from 50 metabolic pathways were identified. The most prominent metabolome changes among the study groups were in lysophosphatidylcholine, acyl carnitine, and amino acid profiles. Several pathways were found perturbed according to the integrated pathway analysis. These pathways ranged from amino acid metabolism (such as tryptophan metabolism) to fatty acid metabolism, namely linoleic and butyrate. These metabolic changes during UC reflect the homeostatic disturbance in the gut, and highlight the importance of system biology approaches to identify key drivers of pathogenesis which prerequisite personalized medicine.
    Keywords:  fatty acid metabolism; inflammatory bowel disease; metabolomics; pathway analysis; personalized treatment; tryptophan metabolism; ulcerative colitis
  5. J Hepatol. 2019 Nov 25. pii: S0168-8278(19)30696-8. [Epub ahead of print]
      BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. Therefore, we aimed to analyze blood metabolome in patients with cirrhosis, with and without ACLF.METHODS: We performed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 650 patients with AD (acute decompensation of cirrhosis, without ACLF), 181 with ACLF, 43 with compensated cirrhosis, and 29 healthy subjects.
    RESULTS: Of the 137 annotated metabolites identified, 100 were increased in patients with ACLF of any grade, relative to those with AD, and 38 composed a distinctive blood metabolite fingerprint for ACLF. Among patients with ACLF, the intensity of the fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that the fingerprint reflected not only decreased kidney excretion but also altered cell metabolism. The higher the ACLF-associated fingerprint intensity, the higher plasma levels of inflammatory markers, tumor necrosis factor α, soluble CD206, and soluble CD163. ACLF was characterized by intense proteolysis and lipolysis; amino acid catabolism; extra-mitochondrial glucose metabolism through glycolysis, pentose phosphate, and D-glucuronate pathways; depressed mitochondrial ATP-producing fatty acid β-oxidation; and extra-mitochondrial amino acid metabolism giving rise to metabolites which are metabotoxins.
    CONCLUSIONS: In ACLF, intense systemic inflammation is associated with blood metabolite accumulation witnessing profound alterations in major metabolic pathways, in particular inhibition in mitochondrial energy production, which may contribute to the existence of organ failures.
    Keywords:  Biomarkers; CANONIC study; Lipidomics; Multiorgan failure; Small-molecules
  6. Physiol Rep. 2019 Nov;7(22): e14275
      Fatty acid (FA)-derived lipid products generated by cytochrome P450 (CYP), lipoxygenase (LOX), and cyclo-oxygenase (COX) influence cardiovascular function. However, plasma measurements invariably ignore 40% of the blood specimen, namely the erythrocytes. These red blood cells (RBCs) represent a cell mass of about 3 kg. RBCs are a potential reservoir for epoxy fatty acids, which on release could regulate vascular capacity. We tested the hypothesis that maximal physical activity would influence the epoxy fatty acid status in RBCs. We used a standardized maximal treadmill exercise according to Bruce to ensure a robust hemodynamic and metabolic response. Central hemodynamic monitoring was performed using blood pressure and heart rate measurements and maximal workload was assessed in metabolic equivalents (METs). We used tandem mass spectrometry (LC-MS/MS) to measure epoxides derived from CYP monooxygenase, as well as metabolites derived from LOX, COX, and CYP hydroxylase pathways. Venous blood was obtained for RBC lipidomics. With the incremental exercise test, increases in the levels of various CYP epoxy-mediators in RBCs, including epoxyoctadecenoic acids (9,10-EpOME, 12,13-EpOME), epoxyeicosatrienoic acids (5,6-EET, 11,12-EET, 14,15-EET), and epoxydocosapentaenoic acids (16,17-EDP, 19,20-EDP) occurred, as heart rate, systolic blood pressure, and plasma lactate concentrations increased. Maximal (13.5 METs) exercise intensity had no effect on diols and various LOX, COX, and hydroxylase mediators. Our findings suggest that CYP epoxy-metabolites could contribute to the cardiovascular response to maximal exercise.
    Keywords:  Exercise; eicosanoids; lipidomics; red blood cells
  7. Methods Mol Biol. 2020 ;2089 251-256
      Lipoxygenases are key enzymes that catalyze the polyunsaturated fatty acids such as arachidic acid, linoleic acid (LA), and others unsaturated fatty acids. They are involved in important functions such as cell structure, metabolism, and signal transduction mechanisms, finally mediating cell death process, especially ferroptosis, a novel type of cell death modality. Our present protocol described a colorimetric assay for measuring lipoxygenase activity as well as a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of arachidonic acid metabolites.
    Keywords:  Arachidic acid; Leukotrienes; Lipoxygenases; Polyunsaturated fatty acids
  8. Sci Rep. 2019 Nov 28. 9(1): 17760
      Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.
  9. Exp Mol Med. 2019 Nov 29. 51(11): 146
      Branched-chain amino acid (BCAA) catabolism and high levels of enzymes in the BCAA metabolic pathway have recently been shown to be associated with cancer growth and survival. However, the precise roles of BCAA metabolism in cancer growth and survival remain largely unclear. Here, we found that BCAA metabolism has an important role in human pancreatic ductal adenocarcinoma (PDAC) growth by regulating lipogenesis. Compared with nontransformed human pancreatic ductal (HPDE) cells, PDAC cells exhibited significantly elevated BCAA uptake through solute carrier transporters, which were highly upregulated in pancreatic tumor tissues compared with normal tissues. Branched-chain amino-acid transaminase 2 (BCAT2) knockdown markedly impaired PDAC cell proliferation, but not HPDE cell proliferation, without significant alterations in glutamate or reactive oxygen species levels. Furthermore, PDAC cell proliferation, but not HPDE cell proliferation, was substantially inhibited upon knockdown of branched-chain α-keto acid dehydrogenase a (BCKDHA). Interestingly, BCKDHA knockdown had no significant effect on mitochondrial metabolism; that is, neither the level of tricarboxylic acid cycle intermediates nor the oxygen consumption rate was affected. However, BCKDHA knockdown significantly inhibited fatty-acid synthesis, indicating that PDAC cells may utilize BCAAs as a carbon source for fatty-acid biosynthesis. Overall, our findings show that the BCAA metabolic pathway may provide a novel therapeutic target for pancreatic cancer.
  10. Mol Metab. 2019 Dec;pii: S2212-8778(19)30907-X. [Epub ahead of print]30 61-71
      OBJECTIVE: The dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways.METHODS: We designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS).
    RESULTS: We show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses.
    CONCLUSIONS: Our data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.
    Keywords:  Acetyl-CoA; Compartmentalization; Mevalonate pathway; Organelle; Stable isotope tracing; Sub-cellular metabolism
  11. Biomed Res Int. 2019 ;2019 3015150
      Cold atmospheric plasma (CAP) is a novel technology, which has been widely applied in biomedicine, especially in wound healing, dermatological treatment, hemostasis, and cancer treatment. In most cases, CAP treatment will interact with innumerable blood capillaries. Therefore, it is important and necessary to understand the effects of CAP treatment on endothelial cell metabolism. In this study, the metabolite profiling of plasma treatment on endothelial cells was measured by gas chromatography tandem time-of-flight mass spectrometry (GC-TOF-MS). We found that 695 signals (metabolites) were detected by GC-TOF-MS and then evaluated using orthogonal projections to latent structures discriminant analysis (OPLS-DA). All the differential metabolites were listed, and proline and xanthosine were the two of the most downregulated metabolites by plasma treatment. By comprehensive metabolic pathway analysis with the KEGG pathway, we showed that alanine, aspartate, glutamate, and purine metabolism pathways were the most significantly suppressed after gas plasma treatment in human endothelial cells. Our finding gives an overall picture of the metabolic pathways affected by plasma treatment in endothelial cells.
  12. Cancers (Basel). 2019 Nov 26. pii: E1870. [Epub ahead of print]11(12):
      This review highlights recent advances in the understanding of the relevance of altered lipid metabolic pathways contributing to the poor prognosis of high grade serous ovarian cancer, as they relate to cancer metastasis and cancer stemness. Increased lipid uptake regulated by the receptor CD36 and the transport protein FABP4 has been implicated in ovarian cancer metastasis. The symbiotic relationship between ovarian cancer cells and adipocytes was shown to be important for sustaining widespread peritoneal and omental metastasis. Increased lipogenesis dependent on the fatty acid desaturase SCD1 was detected in ovarian cancer stem cells. Furthermore, response to therapy, specifically to platinum, was linked to increased fatty acid biogenesis, while the survival of drug tolerant cells was shown to depend on lipid peroxidation. These recent findings suggest that lipids are necessary elements supporting oncogenic signaling and the energetic needs of rapidly proliferating cancer cells. New strategies targeting key enzymes involved in lipid uptake or utilization in cancer cells have been shown to exert anti-tumor effects and are being developed as cancer interventions in combination with chemotherapy or immunotherapy.
    Keywords:  lipid metabolism; ovarian cancer; stem cell
  13. Metabolites. 2019 Nov 26. pii: E289. [Epub ahead of print]9(12):
      Routine diagnostic screening of inborn errors of metabolism (IEM) is currently performed by different targeted analyses of known biomarkers. This approach is time-consuming, targets a limited number of biomarkers and will not identify new biomarkers. Untargeted metabolomics generates a global metabolic phenotype and has the potential to overcome these issues. We describe a novel, single platform, untargeted metabolomics method for screening IEM, combining semi-automatic sample preparation with pentafluorophenylpropyl phase (PFPP)-based UHPLC- Orbitrap-MS. We evaluated analytical performance and diagnostic capability of the method by analysing plasma samples of 260 controls and 53 patients with 33 distinct IEM. Analytical reproducibility was excellent, with peak area variation coefficients below 20% for the majority of the metabolites. We illustrate that PFPP-based chromatography enhances identification of isomeric compounds. Ranked z-score plots of metabolites annotated in IEM samples were reviewed by two laboratory specialists experienced in biochemical genetics, resulting in the correct diagnosis in 90% of cases. Thus, our untargeted metabolomics platform is robust and differentiates metabolite patterns of different IEMs from those of controls. We envision that the current approach to diagnose IEM, using numerous tests, will eventually be replaced by untargeted metabolomics methods, which also have the potential to discover novel biomarkers and assist in interpretation of genetic data.
    Keywords:  HRAM-MS; IEM; LC-MS; Orbitrap; PFPP; PKU; inborn errors of metabolism; metabolomics; organic aciduria; urea cycle defects
  14. J Biol Chem. 2019 Nov 27. pii: jbc.RA119.010032. [Epub ahead of print]
      The SIN3 histone-modifying complex regulates the expression of multiple methionine catabolic genes, including SAM synthetase (Sam-S), as well as S-adenosyl-methionine (SAM) levels. To further dissect the relationship between methionine catabolism and epigenetic regulation by SIN3, we sought to identify genes and metabolic pathways controlled by SIN3 and SAM-S in Drosophila melanogaster. Using several approaches, including RNAi-mediated gene silencing, RNA-seq- and quantitative RT-PCR-based transcriptomics, and ultra-high performance LC-MS/MS- and GC/MS- based metabolomics, we found that as a global transcriptional regulator, SIN3 impacted a wide range of genes and pathways. In contrast, SAM-S affected only a narrow range of genes and pathways. The expression and levels of additional genes and metabolites, however, were altered in Sin3A+Sam-S dual knockdown cells. This analysis revealed that SIN3 and SAM-S regulate overlapping pathways, many of which involve one-carbon and central carbon metabolisms. In some cases, the factors acted independently; in some others, redundantly; and for a third set, in opposition. Together, these results obtained from experiments with the chromatin regulator SIN3 and the metabolic enzyme SAM-S, uncover a complex relationship between metabolism and epigenetic regulation.
    Keywords:  SAM synthetase; SIN3; chromatin; epigenetics; gene regulation; glycolysis; histone modification; metabolism; transcription; tricarboxylic acid cycle (TCA cycle) (Krebs cycle)
  15. J Cancer. 2019 ;10(25): 6395-6404
      Protein kinase D (PKD) has been implicated in cancer cell survival, proliferation, migration and angiogenesis. However, it is still unknown whether PKD regulates cell proliferation through lipid metabolism in cancer cells. Here we report a novel function of PKD3, a member of PKD family, in regulating of prostate cancer cell proliferation by modulation of SREBP1-mediated de novo lipogenesis. We show that silencing of PKD3 significantly reduces lipid content and expression of the lipogenic genes encoding FASN and ATP-citrate lyase (ACLY). Moreover, endogenous PKD3 interacts with sterol regulatory element binding protein 1(SREBP1) in DU145 cells. Interestingly, PKD3 silencing decreases not only the level of matured-SREBP1 (68KD) but also the binding of SREBP1 to the promoter of fasn gene. In addition, overexpression of SREBP1 reverses the suppression of cell growth caused by PKD3 depletion. Finally, immune-histochemical staining indicate that PKD3 expression is positively correlated with expression of FASN and SREBP1 in prostate cancers. Taken together, these data suggest that targeting PKD3-mediated de novo lipogenesis may be a potential therapeutic approach to block prostate cancer progression.
    Keywords:  SREBP1; fatty acid synthesis; lipid metabolism; prostate cancer; protein kinase D3
  16. Anal Chim Acta. 2020 Jan 15. pii: S0003-2670(19)31201-2. [Epub ahead of print]1094 57-69
      In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50-500 Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13 ± 7, 15 ± 9, and 0.6 ± 0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (n = 8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.
    Keywords:  Intercellular platelet signaling; Lipid mediators; Lipidomics; Oxylipins; Platelet activation; SWATH
  17. Int J Mol Sci. 2019 Nov 27. pii: E5957. [Epub ahead of print]20(23):
      A comparative study of the impact of n-butylamine and traditionally used additives (ammonium hydroxide and formic acid) on the efficiency of the electrospray ionization (ESI) process for the enhancement of metabolite coverage was performed by direct injection mass spectrometry (MS) analysis in negative mode. Evaluation of obtained MS data showed that n-butylamine is one of the most effective additives for the analysis of metabolite composition in ESI in negative ion mode (ESI(-)) The limitations of the use of n-butylamine and other alkylamines in the analysis of metabolic composition and a decontamination procedure that can reduce MS device contamination after their application are discussed. The proposed procedure allows the performance of high-sensitivity analysis of low-molecular-weight compounds on the same MS device in both polarities.
    Keywords:  alkylamines; decontamination procedure; metabolome profiling
  18. Neoplasia. 2019 Nov 22. pii: S1476-5586(19)30160-5. [Epub ahead of print]22(1): 22-32
      The microenvironment of solid tumors is a key determinant of therapy efficacy. The co-occurrence of oxygen and nutrient deprivation is a common phenomenon of the tumor microenvironment and associated with treatment resistance. Cholangiocarcinoma (CCA) is characterized by a very poor prognosis and pronounced chemoresistance. A better understanding of the underlying molecular mechanisms is urgently needed to improve therapy strategies against CCA. We sought to investigate the importance of the conditionally essential amino acid glutamine, a centrally important nutrient for a variety of solid tumors, for CCA. Glutamine levels were strongly decreased in CCA samples and the growth of established human CCA cell lines was highly dependent on glutamine. Using gradual reduction of external glutamine, we generated derivatives of CCA cell lines which were able to grow without external glutamine (termed glutamine-depleted (GD)). To analyze the effects of coincident oxygen and glutamine deprivation, GD cells were treated with cisplatin or gemcitabine under normoxia and hypoxia. Strikingly, the well-established phenomenon of hypoxia-induced chemoresistance was completely reversed in GD cells. In order to better understand the underlying mechanisms, we focused on the oncogene c-Myc. The combination of cisplatin and hypoxia led to sustained c-Myc protein expression in wildtype cells. In contrast, c-Myc expression was reduced in response to the combinatorial treatment in GD cells, suggesting a functional importance of c-Myc in the process of hypoxia-induced chemoresistance. In summary, these findings indicate that the mechanisms driving adaption to tumor microenvironmental changes and their relevance for the response to therapy are more complex than expected.
  19. Int J Mol Sci. 2019 Nov 26. pii: E5952. [Epub ahead of print]20(23):
      Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.
    Keywords:  UPLC-ESI-Q-TOF-MS; enterovirus; fatty acid; lipidomics
  20. Anal Chem. 2019 Nov 25.
      Shotgun lipidomics has recently gained popularity for lipid analysis. Conventionally, shotgun analysis of glycerophospholipids via direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides glycerophospholipid (GPL) class (i.e., head group composition) and fatty acyl composition. Reliant on low-energy collision induced dissociation (CID), traditional ESI-MS/MS fails to define fatty acyl regiochemistry along the glycerol backbone or carbon-carbon double bond position(s) in unsaturated fatty acyl substituents. Therefore, isomeric GPLs are often unresolved, representing a significant challenge for shotgun-MS approaches. We developed a top-down shotgun-MS method utilizing gas-phase ion/ion charge inversion chemistry that provides near-complete GPL structural identification. First, in negative ion mode, CID of mass-selected GPL anions generates fatty acyl carboxylate anions via fragmentation of ester bonds linking the fatty acyl substituents at the sn-1 and sn-2 positions of the glycerol backbone. Product anions, including fatty acyl carboxylate ions, were then derivatized in the mass spectrometer via an ion/ion charge inversion reaction with tris-phenanthroline magnesium dications. Subsequent CID of charge inverted fatty acyl complex cations yielded isomer-specific product ion spectra that permit (i) unambiguous assignment of carbon-carbon double bond position(s) and (ii) relative quantitation of isomeric fatty acyl substituents. The outlined strategy was applied to the analysis of targeted GPL extracted from human plasma, including several proposed plasma biomarkers. A single experiment thus facilitates assignment of the GPL head group, fatty acyl composition, carbon-carbon double bond position(s) in unsaturated fatty acyl chains, and, in some cases, fatty acyl sn-position and relative abundances for isomeric fatty acyl substituents. Ultimately, this MSn platform paired with ion/ion chemistry permitted identification of major, and some minor, isomeric contributors that are unresolved using conventional ESI-MS/MS.
  21. J Proteome Res. 2019 Nov 27.
      Whilst many laboratories take appropriate care, there are still cases where the performances of untargeted profiling methods suffer from a lack of design, control and articulation of the various steps involved. This is particularly harmful to modern comprehensive analytical instrumentations that otherwise provide an unprecedented coverage of complex matrices. In this work, we present a global analytical workflow based on comprehensive two-dimensional gas chromatography (GC×GC) coupled to high resolution time-of-flight mass spectrometry (HR-TOF-MS). It was optimized for sample preparation and chromatographic separation, and validated on in-house QC samples and NIST SRM 1950 samples. It also includes a QC procedure, a multi-approaches data (pre)processing workflow and an original bias control procedure. Compounds of interest were identified using mass, retention and biological informations. As a proof of concept, 35 serum samples representing 3 subgroups of Crohn's disease (with high, low and quiescent endoscopic activity) were analyzed along with 33 healthy controls. This led to the selection of 31 unique candidate biomarkers able to classify Crohn's disease and healthy samples with OPLS-DA Q2 0.48 and ROC AUC 0.85 (100% sensitivity and 82% specificity in cross validation). 15 of these 33 candidates were reliably annotated (MSI level 2).
  22. Anal Chem. 2019 Nov 27.
      Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 μM) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.
  23. J Chemother. 2019 Nov 28. 1-11
      Fatty Acid Synthase (FASN) is responsible for the de novo synthesis of fatty acids, which are involved in the preservation of biological membrane structure, energy storage and assembly of factors involved in signal transduction. FASN plays a critical role in supporting tumor cell growth, thus representing a potential target for anti-cancer therapies. Moreover, this enzyme has been recently associated with increased PD-L1 expression, suggesting a role for fatty acids in the impairment of the immune response in the tumor microenvironment. Orlistat, a tetrahydrolipstatin used for the treatment of obesity, has been reported to reduce FASN activity, while inducing a sensible reduction of the growth potential in different cancer models. We have analyzed the effect of orlistat on different features involved in the tumor cell biology of the T-ALL Jurkat cell line. In particular, we have observed that orlistat inhibits Jurkat cell growth and induces a perturbation of cell cycle along with a decline of FASN activity and protein levels. Moreover, the drug produces a remarkable impairment of PD-L1 expression. These findings suggest that orlistat interferes with different mechanisms involved in the control of tumor cell growth and can potentially contribute to decrease the tumor-associated immune-pathogenesis.
    Keywords:  FASN; PD-L1; leukemia
  24. Cancer Res. 2019 Nov 25. pii: canres.0638.2019. [Epub ahead of print]
      Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of glioblastoma metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution which cannot be fully described by limited or uneven disruption of the blood brain barrier. In this work we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of glioblastoma. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were more highly expressed in samples with higher acyl-carnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport, and constitute an important consideration for therapeutic strategies in the treatment of GBM.
  25. Sci Rep. 2019 Nov 29. 9(1): 17978
      Human dermal fibroblasts can be reprogrammed into hepatocyte-like (HEP-L) cells by the expression of a set of transcription factors. Yet, the metabolic rewiring suffered by reprogrammed fibroblasts remains largely unknown. Here we report, using stable isotope-resolved metabolic analysis in combination with metabolomic-lipidomic approaches that HEP-L cells mirrors glutamine/glutamate metabolism in primary cultured human hepatocytes that is very different from parental human fibroblasts. HEP-L cells diverge glutamine from multiple metabolic pathways into deamidation and glutamate secretion, just like periportal hepatocytes do. Exceptionally, glutamine contribution to lipogenic acetyl-CoA through reductive carboxylation is increased in HEP-L cells, recapitulating that of primary cultured human hepatocytes. These changes can be explained by transcriptomic rearrangements of genes involved in glutamine/glutamate metabolism. Although metabolic changes in HEP-L cells are in line with reprogramming towards the hepatocyte lineage, our conclusions are limited by the fact that HEP-L cells generated do not display a complete mature phenotype. Nevertheless, our findings are the first to characterize metabolic adaptation in HEP-L cells that could ultimately be targeted to improve fibroblasts direct reprogramming to HEP-L cells.
  26. Anal Methods. 2019 Jan 07. 11(1): 49-57
      Methylation aberrations play an important role in many metabolic disorders including cancer. Methylated metabolites are direct indicators of metabolic aberrations, and currently, there is no Liquid chromatography - Mass spectrometry (LC-MS) based method available to cover all classes of methylated metabolites at low detection limits. In this study, we have developed a method for the detection of methylated metabolites, and it's biological application. In this approach, we used a HILIC based HPLC with MS to measure methylated organic acids, amino acids, and nucleotides. These metabolites were separated from each other by their hydrophobic interactions and analyzed by targeted metabolomics of single reaction monitoring by positive and negative mode of electrospray ionization. These metabolites were quantified, and the interday reproducibility was <10% relative standard deviation. Furthermore, by applying this method, we identified high levels of methylated metabolites in bladder cancer cell lines compared to benign cells. In vitro treatment of cancer cells with methylation inhibitor, 5- aza-2'-deoxycytidine showed a decrease in these methylated metabolites. This data indicates that HPLC analysis using this HILIC based method could be a powerful tool for measuring methylated metabolites in biological specimens. This method is rapid, sensitive, selective, and precise to measure methylated metabolites.
  27. Anal Bioanal Chem. 2019 Nov 26.
      Insulin oligosaccharide conjugates hold promise as potential glucose-responsive insulins (GRIs), which can improve the therapeutic index of insulins and mitigate the risk of hypoglycemia. A key challenge for the analytical development of such molecules is finding an efficient method to characterize the purity and impurities of conjugated insulins. Using the S-Matrix Fusion QbD-ultrahigh performance liquid chromatography (UHPLC) integrated system, we were able to quickly screen and develop two short UHPLC methods. These methods were used to support process development, clinical batch drug substance (DS) release, and stability studies of MK-2640, an insulin oligosaccharide conjugate. Both methods used a Waters CSH C18 column, with a shallow gradient of acetonitrile to aqueous mobile phase containing 25 mM sodium perchlorate and 0.05% perchloric acid. The 10-min run time method was well suited for process development and monitoring as it was able to separate the main product, MK-2640, six oligosaccharide-substituted recombinant human insulin (RHI) impurities, A21 deamidated MK-2640, and the starting material RHI. The 13-min run time method provided improved separation of the major impurities and demonstrated good chromatographic reproducibility on different instruments or using columns from different lots of stationary phase, which made it ideal for the final DS release. Validation of the 13-min method demonstrated great linearity for both the MK-2640 main peak and its related impurities, low limit of detection (0.02%), and limit of quantitation (0.05%). The high specificity of the method allowed the separation of the degradation products from main peak, thus makes it suitable for stability monitoring. The major impurities in the DS were characterized by two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS).
    Keywords:  2D-LC-MS; Glucose-responsive insulin; Ion pairing; Sodium perchlorate; Ultra-high performance liquid chromatography
  28. Metabolomics. 2019 Nov 26. 15(12): 156
      INTRODUCTION: Diabetes and cancer are among the most frequent causes of death worldwide. Recent epidemiological findings have indicated a link between diabetes and cancer in several organs, particularly the liver. A number of epidemiological studies have demonstrated that diabetes is an established independent risk factor for hepatocellular carcinoma (HCC). However, the metabolites connecting diabetes and HCC remains less well understood.OBJECTIVES: The study aimed to identify clinical and metabolomics differences of HCC from patients with/without diabetes using comprehensive global metabolomics analysis.
    METHODS: Metabolite profiling was conducted with the Metabolon platform for 120 human diabetes/non-diabetes HCC tumor/normal tissues. Standard statistical analyses were performed using the Partek Genomics Suite on log-transformed data. Principal component analysis (PCA) was conducted using all and dysregulated metabolites.
    RESULTS: We identified a group of metabolites that are differentially expressed in the tumor tissues of diabetes HCC compared to non-diabetes HCC patients. Meanwhile, we also identified a group of metabolites that are differentially expressed in the matched normal liver tissues of diabetes HCC compared to non-diabetes HCC patients. Some metabolites are consistently dysregulated in the tumor or matched normal tissues of HCC with or without diabetes. However, some metabolites, including 2-hydroxystearate, were only overexpressed in the tumor tissues of HCC with diabetes and associated with the glucose level.
    CONCLUSION: Metabolic profiling identifies distinct dysregulated metabolites in HCC patients with/without diabetes.
    Keywords:  Diabetes; Hepatocellular carcinoma; Metabolites; Metabolomics; Principal component analysis
  29. Mol Cells. 2019 Nov 30. 42(11): 747-754
      The incidence of atherosclerosis is higher among patients with several autoimmune diseases such as psoriasis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It is well documented that innate immune cells including macrophages and dendritic cells sense lipid species such as saturated fatty acids and oxidized low-density lipoprotein and produce pro-inflammatory cytokines and chemokines. However, whether a hyperlipidemic environment also impacts autoimmune T cell responses has been unclear. Among CD4+ T cells, Th17 and follicular helper T (Tfh) cells are known to play pathogenic roles in the development of hyperlipidemiaassociated autoimmune diseases. This review gives an overview of the cellular and molecular mechanisms by which dysregulated lipid metabolism impacts the pathogenesis of autoimmune diseases, with specific emphasis on Th17 and Tfh cells.
    Keywords:  Tfh cell; Th17 cell; autoimmune diseases; hyperlipidemia; lipid metabolism