bims-mascan 2019-06-30 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–06–30
twelve papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research

Metabolic signatures of cancer cells and stem cells.
Targeting the H3K4 demethylase KDM5B reprograms the metabolome and phenotype of melanoma cells.
Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.
Mass Spectrometry Data Repository Enhances Novel Metabolite Discoveries with Advances in Computational Metabolomics.
Development of a Rapid Ultra High-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Analysis of sn-1 and sn-2 Lysophosphatidic Acid Regioisomers in Mouse Plasma.
Analysis of the Antiproliferative Effect of Ankaferd Hemostat on Caco-2 Colon Cancer Cells via LC/MS Shotgun Proteomics Approach.
Proteomics-Metabolomics Combined Approach Identifies Peroxidasin as a Protector against Metabolic and Oxidative Stress in Prostate Cancer.
Important Metabolites in Maintaining Folate Cycle, Homocysteine, and Polyamine Metabolism Associated with Ranibizumab Treatment in Cultured Human Tenon's Fibroblasts.
UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis.
Identification of a Multiplex Biomarker Panel for Hypertrophic Cardiomyopathy using Quantitative Proteomics and Machine Learning.
Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry.
Development of an IPRP-LC-MS/MS method to determine the fate of intracellular thiamine in cancer cells.

Nat Metab. 2019 Feb;1(2): 177-188

Metabolic signatures of cancer cells and stem cells.

Andrew M Intlekofer, Lydia W S Finley.

In contrast to terminally differentiated cells, cancer cells and stem cells retain the ability to re-enter the cell cycle and proliferate. In order to proliferate, cells must increase the uptake and catabolism of nutrients to support anabolic cell growth. Intermediates of central metabolic pathways have emerged as key players that can influence cell differentiation 'decisions', processes relevant for both oncogenesis and normal development. Consequently, how cells rewire metabolic pathways to support proliferation may have profound consequences for cellular identity. Here, we discuss the metabolic programs that support proliferation and explore how metabolic states are intimately entwined with the cell fate decisions that characterize stem cells and cancer cells. By comparing the metabolism of pluripotent stem cells and cancer cells, we hope to illuminate common metabolic strategies as well as distinct metabolic features that may represent specialized adaptations to unique cellular demands.

DOI: https://doi.org/10.1038/s42255-019-0032-0
J Invest Dermatol. 2019 Jun 20. pii: S0022-202X(19)31782-8. [Epub ahead of print]

Targeting the H3K4 demethylase KDM5B reprograms the metabolome and phenotype of melanoma cells.

Felix C E Vogel, Natalie Bordag, Elmar Zügner, Marija Trajkovic-Arsic, Heike Chauvistré, Batool Shannan, Renáta Váraljai, Susanne Horn, Christoph Magnes, Jens Siveke, Dirk Schadendorf, Alexander Roesch.

Melanoma cells shift between epigenetic-metabolic states to adapt to stress and, particularly, to drugs. Here, we unraveled the metabolome of an H3K4 demethylase (KDM5B/JARID1B)-driven melanoma cell phenotype, which is known to be multi-drug resistant. We set up a fast protocol for standardized, high sensitive liquid-chromatography-high-resolution-mass-spectrometry analyzing stably controlled KDM5B expression by RNAi or doxycycline-induced overexpression. Within the KDM5B-dependent metabolome, we found significant and highly specific regulation of 11 intracellular metabolites. Functionally, overexpression of KDM5B in melanoma cells led to broadening of their oxidative metabolism from mainly glutamine-dependent to additionally glucose- and fatty acid-utilizing, upregulation of the pentose phosphate pathway as a source of anti-oxidant NADPH, and maintenance of a high GSH/GSSG ratio. KDM inhibition (GSK-J1, 2.4-PDCA) decreased colony formation and invasion in 3D models. Thus, targeting KDM5B could represent an alternative way of modulating the metabolome and malignant cell behavior in melanoma.

DOI: https://doi.org/10.1016/j.jid.2019.06.124
J Biol Chem. 2019 Jun 24. pii: jbc.RA119.008680. [Epub ahead of print]

Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.

Yudong Wang, Johan Palmfeldt, Niels Gregersen, Alexander M Makhov, James F Conway, Meicheng Wang, Stephen P McCalley, Shrabani Basu, Hana Alharbi, Claudette St Croix, Michael J Calderon, Simon Watkins, Jerry Vockley.

  Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells - the electron transfer chain (ETC), fatty acid β-oxidation (FAO), and the tricarboxylic acid (TCA) cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase (VLCAD) physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.

Keywords:  electron microscopy (EM); fatty acid oxidation; mitochondrial metabolism; mitochondrial respiratory chain complex; primary metabolism; protein structure; proteomics; stimulated emission depletion microscopy (STED); supercomplex; trifunctional protein (TFP)

DOI:  https://doi.org/10.1074/jbc.RA119.008680
Metabolites. 2019 Jun 24. pii: E119. [Epub ahead of print]9(6):

Mass Spectrometry Data Repository Enhances Novel Metabolite Discoveries with Advances in Computational Metabolomics.

Hiroshi Tsugawa, Aya Satoh, Haruki Uchino, Tomas Cajka, Makoto Arita, Masanori Arita.

  Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.

Keywords:  computational metabolomics; data processing; data repository; lipidomics; reanalysis

DOI:  https://doi.org/10.3390/metabo9060119
Lipids. 2019 Jun 24.

Development of a Rapid Ultra High-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Analysis of sn-1 and sn-2 Lysophosphatidic Acid Regioisomers in Mouse Plasma.

Juan J Aristizabal-Henao, Maria F Fernandes, Robin E Duncan, Ken D Stark.

  Lysophosphatidic acids (lysoPtdOH) are involved in several physiological processes including cell proliferation, inflammation, and glucose metabolism. However, measuring lysoPtdOH is challenging due to inadequate extraction techniques, poor chromatographic resolution, or the inability to discriminate between sn-1 and sn-2 regioisomers. In the present work, we developed a high-throughput (10 min run times) ultra-high-performance liquid chromatography-tandem mass spectrometry method capable of discriminating lysoPtdOH species by their fatty acyl composition and sn-localization on glycerol backbones. We quantitated sn-1/sn-2 regioisomeric pairs of lysoPtdOH with 16:0, 18:0, 18:1, 18:2, 20:4, and 22:6 fatty acyl chains using 50 μL of mouse plasma. The method presented here can be expanded to profile more lysoPtdOH species, and has the potential to be used in clinical settings to quickly screen lysoPtdOH profiles. Finally, the ability to discriminate between sn-1 and sn-2 isomers can provide insights regarding the metabolic origins and fates of specific lysoPtdOH molecules.

Keywords:  Analytical chemistry; HPLC (high performance liquid chromatography); Mass spectrometry; Phospholipid analysis

DOI:  https://doi.org/10.1002/lipd.12172
Biomed Res Int. 2019 ;2019 5268031

Analysis of the Antiproliferative Effect of Ankaferd Hemostat on Caco-2 Colon Cancer Cells via LC/MS Shotgun Proteomics Approach.

Engin Koçak, Mustafa Çelebier, Ibrahim C Haznedaroglu, Sacide Altınöz.

Ankaferd hemostat (ABS), a traditional herbal extract, is a hemostatic agent used for wound healing and bleeding treatment. A standardized form of plants contains many biomolecules. In recent years, previous studies have demonstrated the antineoplastic effect of ABS. In the present work, we focused on the mechanism of its antineoplastic effect over Caco-2 colon cancer cells. The LC/MS-based proteomics method was used to understand the effect of ABS at the protein level. The results were evaluated with gene ontology, protein interaction, and pathway analysis. As shown by our results, ABS altered glucose, fatty acids, and protein metabolism. Moreover, ABS affects the cell cycle machinery. Moreover, we found that ABS induced critical cancer target and suppressor proteins such as carboxyl-terminal hydrolase 1, 60S ribosomal protein L5, Tumor protein D52-like2, karyopherin alpha 2, and protein deglycase DJ-1. In conclusion, the proteomics results indicated that ABS affects various cancer targets and suppressor proteins. Moreover ABS has systematical effect on cell metabolism and cell cycle in Caco-2 cells, suggesting that it could be used as an antineoplastic agent.

DOI: https://doi.org/10.1155/2019/5268031
Int J Mol Sci. 2019 Jun 21. pii: E3046. [Epub ahead of print]20(12):

Proteomics-Metabolomics Combined Approach Identifies Peroxidasin as a Protector against Metabolic and Oxidative Stress in Prostate Cancer.

Jodi Dougan, Ohuod Hawsawi, Liza J Burton, Gabrielle Edwards, Kia Jones, Jin Zou, Peri Nagappan, Guangdi Wang, Qiang Zhang, Alira Danaher, Nathan Bowen, Cimona Hinton, Valerie A Odero-Marah.

  Peroxidasin (PXDN), a human homolog of Drosophila PXDN, belongs to the family of heme peroxidases and has been found to promote oxidative stress in cardiovascular tissue, however, its role in prostate cancer has not been previously elucidated. We hypothesized that PXDN promotes prostate cancer progression via regulation of metabolic and oxidative stress pathways. We analyzed PXDN expression in prostate tissue by immunohistochemistry and found increased PXDN expression with prostate cancer progression as compared to normal tissue or cells. PXDN knockdown followed by proteomic analysis revealed an increase in oxidative stress, mitochondrial dysfunction and gluconeogenesis pathways. Additionally, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics confirmed that PXDN knockdown induced global reprogramming associated with increased oxidative stress and decreased nucleotide biosynthesis. We further demonstrated that PXDN knockdown led to an increase in reactive oxygen species (ROS) associated with decreased cell viability and increased apoptosis. Finally, PXDN knockdown decreased colony formation on soft agar. Overall, the data suggest that PXDN promotes progression of prostate cancer by regulating the metabolome, more specifically, by inhibiting oxidative stress leading to decreased apoptosis. Therefore, PXDN may be a biomarker associated with prostate cancer and a potential therapeutic target.

Keywords:  PXDN; apoptosis; metabolome; oxidative stress; prostate cancer

DOI:  https://doi.org/10.3390/ijms20123046
Biomolecules. 2019 Jun 22. pii: E243. [Epub ahead of print]9(6):

Important Metabolites in Maintaining Folate Cycle, Homocysteine, and Polyamine Metabolism Associated with Ranibizumab Treatment in Cultured Human Tenon's Fibroblasts.

Siti Munirah Md Noh, Siti Hamimah Sheikh Abdul Kadir, Sushil Vasudevan.

  The anti-fibrotic properties of ranibizumab have been well documented. As an antagonist to vascular endothelial growth factor (VEGF), ranibizumab works by binding and neutralizing all active VEGF-A, thus limiting progressive cell growth and proliferation. Ranibizumab application in ocular diseases has shown remarkable desired effects; however, to date, its antifibrotic mechanism is not well understood. In this study, we identified metabolic changes in ranibizumab-treated human Tenon's fibroblasts (HTFs). Cultured HTFs were treated for 48 h with 0.5 mg/mL of ranibizumab and 0.5 mg/mL control IgG antibody which serves as a negative control. Samples from each group were injected into Agilent 6520 Q-TOF liquid chromatography/mass spectrometer (LC/MS) system to establish the metabolite expression in both ranibizumab treated cells and control group. Data obtained was analyzed using Agilent Mass Hunter Qualitative Analysis software to identify the most regulated metabolite following ranibizumab treatment. At p-value < 0.01 with the cut off value of two-fold change, 31 identified metabolites were found to be significantly upregulated in ranibizumab-treated group, with six of the mostly upregulated having insignificant role in fibroblast cell cycle and wound healing regulations. Meanwhile, 121 identified metabolites that were downregulated, and seven of the mostly downregulated are significantly involved in cell cycle and proliferation. Our findings suggest that ranibizumab abrogates the tissue scarring and wound healing process by regulating the expression of metabolites associated with fibrotic activity. In particular, we found that vitamin Bs are important in maintaining normal folate cycle, nucleotide synthesis, and homocysteine and spermidine metabolism. This study provides an insight into ranibizumab's mechanism of action in HTFs from the perspective of metabolomics.

Keywords:  anti-VEGF; ranibizumab; trabeculectomy

DOI:  https://doi.org/10.3390/biom9060243
Nature. 2019 Jun 26.

UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis.

Xiongjun Wang, Ruilong Liu, Wencheng Zhu, Huiying Chu, Hua Yu, Ping Wei, Xueyuan Wu, Hongwen Zhu, Hong Gao, Ji Liang, Guohui Li, Weiwei Yang.

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths1. Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival2,3. However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid4. Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial-mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA.

DOI: https://doi.org/10.1038/s41586-019-1340-y
Mol Cell Proteomics. 2019 Jun 26. pii: mcp.RA119.001586. [Epub ahead of print]

Identification of a Multiplex Biomarker Panel for Hypertrophic Cardiomyopathy using Quantitative Proteomics and Machine Learning.

Gabriella Captur, Wendy E Heywood, Caroline Coats, Stefania Rosmini, Vimal Patel, Luis R Lopes, Richard Collis, Nina Patel, Petros Syrris, Paul Bassett, Ben O'Brien, James Moon, Perry M Elliott, Kevin Mills.

  Hypertrophic cardiomyopathy (HCM) is defined by pathological left ventricular hypertrophy (LVH). It is the commonest inherited cardiac condition and a significant number of high risk cases still go undetected until a sudden cardiac death (SCD) event. Plasma biomarkers do not currently feature in the assessment of HCM disease progression, which is tracked by serial imaging, or in SCD risk stratification which is based on imaging parameters and patient/family history. There is a need for new HCM plasma biomarkers to refine disease monitoring and improve patient risk stratification. To identify new plasma biomarkers for patients with HCM, we performed exploratory myocardial and plasma proteomics screens and subsequently developed a multiplexed targeted liquid chromatography-tandem/mass spectrometry-based assay to validate the 26 peptide biomarkers that were identified. The association of discovered biomarkers with clinical phenotypes was prospectively tested in plasma from 110 HCM patients with LVH (LVH+ HCM), 97 controls and 16 HCM sarcomere gene mutation carriers before the development of LVH (subclinical HCM). Six peptides (Aldolase Fructose-Bisphosphate A, Complement C3, Glutathione S-Transferase Omega 1, Ras Suppressor Protein 1, Talin 1, and Thrombospondin 1) were increased significantly in the plasma of LVH+ HCM compared to controls and correlated with imaging markers of phenotype severity: LV wall thickness, mass and % myocardial scar on cardiovascular magnetic resonance imaging. Using supervised machine learning, this six-biomarker panel differentiated between LVH+ HCM and controls, with an area under the curve of ≥0.87. Five of these peptides were also significantly increased in subclinical HCM compared to controls. In LVH+ HCM, the 6-marker panel correlated with the presence of non-sustained ventricular tachycardia and the estimated 5-year risk of sudden cardiac death. Using quantitative proteomic approaches, we have discovered six potentially useful circulating plasma biomarkers related to myocardial substrate changes in HCM, which correlate with the estimated sudden cardiac death risk.

Keywords:  Cardiovascular disease; Cardiovascular function or biology; Diagnostic; Mass Spectrometry; Multiple reaction monitoring

DOI:  https://doi.org/10.1074/mcp.RA119.001586
J Vis Exp. 2019 Jun 09.

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry.

Ami Maruyama, Kenjiro Kami, Kazunori Sasaki, Hajime Sato, Yuzo Sato, Katsuya Tsuchihara, Hideki Makinoshima.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

DOI: https://doi.org/10.3791/59551
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jun 05. pii: S1570-0232(18)31816-6. [Epub ahead of print]1124 247-255

Development of an IPRP-LC-MS/MS method to determine the fate of intracellular thiamine in cancer cells.

Jaeah Kim, Hunter C Jonus, Jason A Zastre, Michael G Bartlett.

  Understanding the mechanisms underlying cancer cell survival is critical toward advancing drug discovery efforts in this field. Supplemental vitamins have been proposed to play a role in cancer cell metabolism because the increased supply of nutrients is thought to provide cofactors supporting the higher metabolic rate of cancer cells. Particularly, the role of thiamine (vitamin B1) in many biochemical pathways that supports cancer cell metabolism has been investigated. Consequently, the analysis of thiamine and its derivatives in a manner that reflects its dynamic response to genetic modification and pathophysiological stimuli is essential. In this work, we developed a mass spectrometry based-analytical method to track metabolites derived from stable isotope tracers for a better understanding of the metabolic fate of thiamine in cancer cells. This method used ion-pair reversed phase liquid chromatography to simultaneously quantify underivatized thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cells. Hexylamine was used as an ion-pairing agent. The method was successfully validated for accuracy, precision and selectivity in accordance with U.S. FDA guidance. Furthermore, the method was then applied for the determination of thiamine and its derivatives with stable isotope labeling to explore the metabolic fate of intracellular thiamine in cancer cells. The finding shows that thiamine is rapidly converted to TPP however, the TPP does not return to thiamine. It appears that TPP may be utilized for other purposes rather than simply being an enzyme cofactor, suggesting unexplored roles for thiamine in cancer.

Keywords:  Hexylamine; Liquid chromatography-mass spectrometry; Stable isotope-labeled thiamine; Thiamine; Thiamine kinetics; Thiamine pyrophosphate

DOI:  https://doi.org/10.1016/j.jchromb.2019.05.037