bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒04‒28
fifty-three papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. Nature. 2019 Apr 24.
    Chowdhry S, Zanca C, Rajkumar U, Koga T, Diao Y, Raviram R, Liu F, Turner K, Yang H, Brunk E, Bi J, Furnari F, Bafna V, Ren B, Mischel PS.
      Precision oncology hinges on linking tumour genotype with molecularly targeted drugs1; however, targeting the frequently dysregulated metabolic landscape of cancer has proven to be a major challenge2. Here we show that tissue context is the major determinant of dependence on the nicotinamide adenine dinucleotide (NAD) metabolic pathway in cancer. By analysing more than 7,000 tumours and 2,600 matched normal samples of 19 tissue types, coupled with mathematical modelling and extensive in vitro and in vivo analyses, we identify a simple and actionable set of 'rules'. If the rate-limiting enzyme of de novo NAD synthesis, NAPRT, is highly expressed in a normal tissue type, cancers that arise from that tissue will have a high frequency of NAPRT amplification and be completely and irreversibly dependent on NAPRT for survival. By contrast, tumours that arise from normal tissues that do not express NAPRT highly are entirely dependent on the NAD salvage pathway for survival. We identify the previously unknown enhancer that underlies this dependence. Amplification of NAPRT is shown to generate a pharmacologically actionable tumour cell dependence for survival. Dependence on another rate-limiting enzyme of the NAD synthesis pathway, NAMPT, as a result of enhancer remodelling is subject to resistance by NMRK1-dependent synthesis of NAD. These results identify a central role for tissue context in determining the choice of NAD biosynthetic pathway, explain the failure of NAMPT inhibitors, and pave the way for more effective treatments.
    DOI:  https://doi.org/10.1038/s41586-019-1150-2
  2. Life Sci. 2019 Apr 23. pii: S0024-3205(19)30315-7. [Epub ahead of print]
    Hussain M, Adah D, Tariq M, Lu Y, Zhang J, Liu J.
      The tumor microenvironment comprises stromal and tumor cells which interact with each other through complex cross-talks that are mediated by a variety of growth factors, cytokines, and chemokines. The chemokine ligand 13 (CXCL13) and its chemokine receptor 5 (CXCR5) are among the key chemotactic factors which play crucial roles in deriving cancer cell biology. CXCL13/CXCR5 signaling axis makes pivotal contributions to the development and progression of several human cancers. In this review, we discuss how CXCL13/CXCR5 signaling modulates cancer cell ability to grow, proliferate, invade, and metastasize. Furthermore, we also discuss the preliminary evidence on context-dependent functioning of this axis within the tumor-immune microenvironment, thus, highlighting its potential dichotomy with respect to anticancer immunity and cancer immune-evasion mechanisms. At the end, we briefly shed light on the therapeutic potential or implications of targeting CXCL13/CXCR5 axis within the tumor microenvironment.
    Keywords:  CXCL13; CXCR5; Cancer; Immune-evasion; Tumor immunity; Tumor progression
    DOI:  https://doi.org/10.1016/j.lfs.2019.04.053
  3. Int J Mol Sci. 2019 Apr 19. pii: E1933. [Epub ahead of print]20(8):
    Sousa B, Pereira J, Paredes J.
      Cancer cells preferentially use aerobic glycolysis over mitochondria oxidative phosphorylation for energy production, and this metabolic reprogramming is currently recognized as a hallmark of cancer. Oncogenic signaling frequently converges with this metabolic shift, increasing cancer cells' ability to produce building blocks and energy, as well as to maintain redox homeostasis. Alterations in cell-cell and cell-extracellular matrix (ECM) adhesion promote cancer cell invasion, intravasation, anchorage-independent survival in circulation, and extravasation, as well as homing in a distant organ. Importantly, during this multi-step metastatic process, cells need to induce metabolic rewiring, in order to produce the energy needed, as well as to impair oxidative stress. Although the individual implications of adhesion molecules and metabolic reprogramming in cancer have been widely explored over the years, the crosstalk between cell adhesion molecular machinery and metabolic pathways is far from being clearly understood, in both normal and cancer contexts. This review summarizes our understanding about the influence of cell-cell and cell-matrix adhesion in the metabolic behavior of cancer cells, with a special focus concerning the role of classical cadherins, such as Epithelial (E)-cadherin and Placental (P)-cadherin.
    Keywords:  ECM; adhesion; cadherin; cancer; cancer stem cells; metabolism
    DOI:  https://doi.org/10.3390/ijms20081933
  4. Biosci Rep. 2019 Apr 26. pii: BSR20181601. [Epub ahead of print]
    Johnston K, Kim DH, Kerkhoven EJ, Burchmore RJS, Barrett M, Achcar F.
      The metabolism of the parasite Trypanosoma brucei has been the focus of numerous studies since the 1940's. Recently it was shown, using metabolomics coupled with heavy-atom isotope-labelled glucose, that the metabolism of the bloodstream form parasite is more complex than previously thought. This study also raised a number of questions regarding the origin of several metabolites, for example succinate, only a proportion of which derives from glucose. In order to answer some of these questions and explore the metabolism of bloodstream form T. brucei in more depth we followed the fate of five heavy-labelled amino acids - glutamine, proline, methionine, cysteine and arginine - using an LC-MS based metabolomics approach. We found that some of these amino acids have roles beyond those previously thought and we have tentatively identified some unexpected metabolites which need to be confirmed and their function determined.
    Keywords:  amino acid metabolism; mass spectrometry; parasitic protozoa; trypanosomes
    DOI:  https://doi.org/10.1042/BSR20181601
  5. Front Oncol. 2019 ;9 218
    Schwartsburd P.
      Unrestricted cancer growth requires permanent supply of glucose that can be obtained from cancer-mediated reprogramming of glucose metabolism in the cancer-bearing host. The pathological mechanisms by which cancer cells exert their negative influence on host glucose metabolism are largely unknown. This paper proposes a mechanism of metabolic and hormonal changes that may favor glucose delivery to tumor (not host) cells by creating a cancer-host "vicious cycle" whose prolonged action drives cancer progression and promotes host cachexia. To verify this hypothesis, a feedback model of host-cancer interactions that create the "vicious cycle" via cancer-induced reprogramming of host glucose metabolism is proposed. This model is capable of answering some crucial questions as to how anabolic cancer cells can reprogram the systemic glucose metabolism and why these pathways were not observed in pregnancy. The current paper helps to better understanding a pathogenesis of cancer progression and identify hormonal/metabolic targets for anti-cancer treatment.
    Keywords:  cancer biology; glucose; insulin resistance; metabolism; pregnancy; stress
    DOI:  https://doi.org/10.3389/fonc.2019.00218
  6. Nat Commun. 2019 Apr 23. 10(1): 1841
    Ortmayr K, Dubuis S, Zampieri M.
      Transcriptional reprogramming of cellular metabolism is a hallmark of cancer. However, systematic approaches to study the role of transcriptional regulators (TRs) in mediating cancer metabolic rewiring are missing. Here, we chart a genome-scale map of TR-metabolite associations in human cells using a combined computational-experimental framework for large-scale metabolic profiling of adherent cell lines. By integrating intracellular metabolic profiles of 54 cancer cell lines with transcriptomic and proteomic data, we unraveled a large space of associations between TRs and metabolic pathways. We found a global regulatory signature coordinating glucose- and one-carbon metabolism, suggesting that regulation of carbon metabolism in cancer may be more diverse and flexible than previously appreciated. Here, we demonstrate how this TR-metabolite map can serve as a resource to predict TRs potentially responsible for metabolic transformation in patient-derived tumor samples, opening new opportunities in understanding disease etiology, selecting therapeutic treatments and in designing modulators of cancer-related TRs.
    DOI:  https://doi.org/10.1038/s41467-019-09695-9
  7. Cancer Discov. 2019 Apr 26.
      Fatty acid transport protein 2 is required for the immunosuppressive activity of PMN-MDSCs.
    DOI:  https://doi.org/10.1158/2159-8290.CD-RW2019-064
  8. Mol Cancer Ther. 2019 Apr 26. pii: molcanther.0804.2018. [Epub ahead of print]
    Rice MA, Hsu EC, Aslan M, Ghoochani A, Su A, Stoyanova T.
      Prostate cancer remains among the leading causes of cancer-related deaths in men. Patients with aggressive disease typically undergo hormone-deprivation therapy. While treatment is initially very successful, these men commonly progress to lethal, castration resistant prostate cancer in 2-3 years. Standard therapies for castration resistant prostate cancer include second-generation anti-androgens, which prolong patient lifespan by only several months. It is imperative to advance our understanding of the mechanisms leading to resistance to identify new therapies for aggressive prostate cancer. This study identifies Notch1 as a therapeutic target in prostate cancer. Loss of Notch1 in aggressive prostate cancer cells decreases proliferation, invasion and tumorsphere formation. Therapeutic inhibition of Notch1 activity with gamma secretase inhibitors RO4929097 or DAPT in prostate cancer cells further results in decreased proliferative abilities. Loss of Notch1 and treatment of immunocompromised mice bearing prostate cancer xenografts with RO4929097 display significantly impaired tumor growth. Loss of Notch1 additionally decreased metastatic potential of prostate cancer cells in invasion assays in vitro as well as in vivo experiments. Moreover, treatment with gamma secretase inhibitors, or Notch1 gene deletion synergized with anti-androgen therapies, Enzalutamide or Abiraterone, to decrease the growth of prostate cancer cells. Combination of gamma secretase inhibitors with Abiraterone significantly inhibited cell migration and invasion, while combination with Enzalutamide reversed Enzalutamide induced migration and invasion. These collective findings suggest loss of Notch1 delays growth of CRPC, inhibits metastasis, and inhibition of Notch1 activation in conjunction with second-generation anti-androgen therapies could delay growth and progression of prostate cancer.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-18-0804
  9. J Biol Chem. 2019 Apr 26. 294(17): 6692
    Hansen HS.
      
    DOI:  https://doi.org/10.1074/jbc.L119.008356
  10. Cell Rep. 2019 Apr 23. pii: S2211-1247(19)30451-6. [Epub ahead of print]27(4): 997-1007.e5
    Teo YV, Rattanavirotkul N, Olova N, Salzano A, Quintanilla A, Tarrats N, Kiourtis C, Müller M, Green AR, Adams PD, Acosta JC, Bird TG, Kirschner K, Neretti N, Chandra T.
      Oncogene-induced senescence (OIS) is a tumor suppressive response to oncogene activation that can be transmitted to neighboring cells through secreted factors of the senescence-associated secretory phenotype (SASP). Currently, primary and secondary senescent cells are not considered functionally distinct endpoints. Using single-cell analysis, we observed two distinct transcriptional endpoints, a primary endpoint marked by Ras and a secondary endpoint marked by Notch activation. We find that secondary oncogene-induced senescence in vitro and in vivo requires Notch, rather than SASP alone, as previously thought. Moreover, Notch signaling weakens, but does not abolish, SASP in secondary senescence. Global transcriptomic differences, a blunted SASP response, and the induction of fibrillar collagens in secondary senescence point toward a functional diversification between secondary and primary senescence.
    Keywords:  CEBPB; Notch; TGFB; bystander senescence; oncogene induced senescence; paracrine senescence; secondary senescence; senescence; senescence associated secretory phenotype; single-cell RNA sequencing
    DOI:  https://doi.org/10.1016/j.celrep.2019.03.104
  11. Prostate. 2019 Apr 24.
    Duijvesz D, Rodriguez-Blanco G, Hoogland AM, Verhoef EI, Dekker LJ, Roobol MJ, van Leenders GJLH, Luider TM, Jenster G.
      BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value.METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome.
    RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009).
    CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.
    Keywords:  PDCD6IP; XPO1; biomarker; extracellular vesicles; prostate cancer; tissue microarray
    DOI:  https://doi.org/10.1002/pros.23813
  12. Int J Mol Sci. 2019 Apr 12. pii: E1813. [Epub ahead of print]20(8):
    Kohaar I, Petrovics G, Srivastava S.
      Prostate cancer is the most prevalent non-skin cancer in men and is the leading cause of cancer-related death. Early detection of prostate cancer is largely determined by a widely used prostate specific antigen (PSA) blood test and biopsy is performed for definitive diagnosis. Prostate cancer is asymptomatic in the early stage of the disease, comprises of diverse clinico-pathologic and progression features, and is characterized by a large subset of the indolent cancer type. Therefore, it is critical to develop an individualized approach for early detection, disease stratification (indolent vs. aggressive), and prediction of treatment response for prostate cancer. There has been remarkable progress in prostate cancer biomarker discovery, largely through advancements in genomic technologies. A rich array of prostate cancer diagnostic and prognostic tests has emerged for serum (4K, phi), urine (Progensa, T2-ERG, ExoDx, SelectMDx), and tumor tissue (ConfirmMDx, Prolaris, Oncoytype DX, Decipher). The development of these assays has created new opportunities for improving prostate cancer diagnosis, prognosis, and treatment decisions. While opening exciting opportunities, these developments also pose unique challenges in terms of selecting and incorporating these assays into the continuum of prostate cancer patient care.
    Keywords:  diagnosis; molecular biomarkers; prognosis; prostate cancer
    DOI:  https://doi.org/10.3390/ijms20081813
  13. J Pharm Biomed Anal. 2019 Apr 15. pii: S0731-7085(18)32405-1. [Epub ahead of print]172 26-32
    Christner SM, Parise RA, Ivy PS, Tawbi H, Chu E, Beumer JH.
      We have developed a high performance liquid chromatography mass spectrometry method for quantitating paclitaxel and its 6-alpha-OH and 3-para-OH metabolites in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 μm, 2 mm × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000Q with electrospray, positive-mode ionization. The assay was linear from 10-10,000 ng/mL for paclitaxel and 1-1000 ng/mL for both metabolites and proved to be accurate (94.3-110.4%) and precise (<11.3%CV). Recovery from plasma was 59.3-91.3% and matrix effect was negligible (-3.5 to 6.2%). Plasma freeze thaw stability (90.2-107.0%), stability for 37 months at -80 °C (89.4-112.6%), and stability for 4 h at room temperature (87.7-100.0%) were all acceptable. This assay will be an essential tool to further define the metabolism and pharmacology of paclitaxel and metabolites in the clinical setting. The assay may be utilized for therapeutic drug monitoring of paclitaxel and may also reveal the CYP2C8 and CYP3A4 activity phenotype of patients.
    Keywords:  Assay; Metabolites; Paclitaxel; Tandem mass spectrometry; Validation
    DOI:  https://doi.org/10.1016/j.jpba.2019.04.027
  14. Methods Mol Biol. 2019 ;1981 15-23
    Dewaele D, Annaert P, Hoeben E.
      Over the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the quantification of bile acids (BA) and their conjugates in different matrices, such as plasma, blood, urine, and cell lysates. Numerous reports have indeed been published describing methods for quantitative determination of bile acids in plasma samples obtained during in vivo studies. However, information on bioanalytical methods suitable for determination of bile acids in in vitro samples remained scarce. Therefore, we presently report a simple and accurate LC-MS/MS method for the quantification of BA in cells (e.g., cultured human hepatocytes) and corresponding cell culture medium, obtained during in vitro experiments.
    Keywords:  Bile acids; Bioanalysis; Cell culture; Electrospray ionization (ESI); Hepatocytes; In vitro assay; Internal standard (IS); Liquid chromatography (LC); Quantification; Tandem mass spectrometry (MS/MS)
    DOI:  https://doi.org/10.1007/978-1-4939-9420-5_2
  15. J Cancer Metastasis Treat. 2019 ;pii: 26. [Epub ahead of print]5
    Rizzieri D, Paul B, Kang Y.
      Metabolism is defined as the collection of complex biochemical processes that living cells use to generate energy and maintain their growth and survival. Metabolism encompasses the synthesis and breakdown of glucose, fatty acids, and amino acids; the generation of energy (ATP); and oxidative phosphorylation. In cancer cells, metabolism can be commandeered to promote tumor growth and cellular proliferation. These alterations in metabolism have emerged as an additional hallmark of various cancers. In this review we focus on metabolic alterations in multiple myeloma (MM) - a malignancy of plasma cells - including derangements in glycolysis, gluconeogenesis, the tricarboxylic acid cycle, oxidative phosphorylation, and fatty acid/amino acid synthesis and degradation. Particular focus is given to metabolic alterations that contribute to myeloma cell growth, proliferation and drug resistance. Finally, novel approaches that target metabolic pathways for the treatment of MM are discussed.
    Keywords:  Metabolism; alterations; multiple myeloma; treatment
    DOI:  https://doi.org/10.20517/2394-4722.2019.05
  16. Clin Proteomics. 2019 ;16 15
    Zhou B, Yan Y, Wang Y, You S, Freeman MR, Yang W.
      Background: Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases.Methods: By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens.
    Results: Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM (v3.0), a high-quality collection of 4274 experimentally verified mammalian protein complexes. Differential expression and gene ontology (GO) enrichment analyses suggested that 13 integrin complexes involved in cell adhesion were significantly downregulated in both low- and high-grade PCa compared with normal prostate, and that four Prothymosin alpha (ProTα) complexes were significantly upregulated in high-grade PCa compared with normal prostate. Moreover, differential co-regulation and GO enrichment analyses indicated that the assembly levels of six protein complexes involved in RNA splicing were significantly increased in low-grade PCa, and those of four subcomplexes of mitochondrial complex I were significantly increased in high-grade PCa, compared with normal prostate.
    Conclusions: In summary, to the best of our knowledge, the study represents the first large-scale and quantitative, albeit indirect, comparison of individual protein complexes in human PCa tissue specimens. It may serve as a useful resource for better understanding the deregulation of protein complexes in primary PCa.
    Keywords:  Differential co-regulation analysis; Differential expression analysis; Prostate cancer; Protein complex; Quantitative proteomics; TMT-SPS-MS3; Tissue
    DOI:  https://doi.org/10.1186/s12014-019-9236-2
  17. Nucleic Acids Res. 2019 Apr 27. pii: gkz299. [Epub ahead of print]
    Gabriels R, Martens L, Degroeve S.
      MS²PIP is a data-driven tool that accurately predicts peak intensities for a given peptide's fragmentation mass spectrum. Since the release of the MS²PIP web server in 2015, we have brought significant updates to both the tool and the web server. In addition to the original models for CID and HCD fragmentation, we have added specialized models for the TripleTOF 5600+ mass spectrometer, for TMT-labeled peptides, for iTRAQ-labeled peptides, and for iTRAQ-labeled phosphopeptides. Because the fragmentation pattern is heavily altered in each of these cases, these additional models greatly improve the prediction accuracy for their corresponding data types. We have also substantially reduced the computational resources required to run MS²PIP, and have completely rebuilt the web server, which now allows predictions of up to 100 000 peptide sequences in a single request. The MS²PIP web server is freely available at https://iomics.ugent.be/ms2pip/.
    DOI:  https://doi.org/10.1093/nar/gkz299
  18. Methods Protoc. 2019 Mar;pii: 8. [Epub ahead of print]2(1):
    Meyer JG.
      The identification of nearly all proteins in a biological system using data-dependent acquisition (DDA) tandem mass spectrometry has become routine for organisms with relatively small genomes such as bacteria and yeast. Still, the quantification of the identified proteins may be a complex process and often requires multiple different software packages. In this protocol, I describe a flexible strategy for the identification and label-free quantification of proteins from bottom-up proteomics experiments. This method can be used to quantify all the detectable proteins in any DDA dataset collected with high-resolution precursor scans and may be used to quantify proteome remodeling in response to drug treatment or a gene knockout. Notably, the method is statistically rigorous, uses the latest and fastest freely-available software, and the entire protocol can be completed in a few hours with a small number of data files from the analysis of yeast.
    Keywords:  data-dependent acquisition; mass spectrometry; peptide quantification; protein quantification; shotgun proteomics
    DOI:  https://doi.org/10.3390/mps2010008
  19. Metabolites. 2019 Apr 13. pii: E72. [Epub ahead of print]9(4):
    Djoumbou-Feunang Y, Pon A, Karu N, Zheng J, Li C, Arndt D, Gautam M, Allen F, Wishart DS.
      Metabolite identification for untargeted metabolomics is often hampered by the lack of experimentally collected reference spectra from tandem mass spectrometry (MS/MS). To circumvent this problem, Competitive Fragmentation Modeling-ID (CFM-ID) was developed to accurately predict electrospray ionization-MS/MS (ESI-MS/MS) spectra from chemical structures and to aid in compound identification via MS/MS spectral matching. While earlier versions of CFM-ID performed very well, CFM-ID's performance for predicting the MS/MS spectra of certain classes of compounds, including many lipids, was quite poor. Furthermore, CFM-ID's compound identification capabilities were limited because it did not use experimentally available MS/MS spectra nor did it exploit metadata in its spectral matching algorithm. Here, we describe significant improvements to CFM-ID's performance and speed. These include (1) the implementation of a rule-based fragmentation approach for lipid MS/MS spectral prediction, which greatly improves the speed and accuracy of CFM-ID; (2) the inclusion of experimental MS/MS spectra and other metadata to enhance CFM-ID's compound identification abilities; (3) the development of new scoring functions that improves CFM-ID's accuracy by 21.1%; and (4) the implementation of a chemical classification algorithm that correctly classifies unknown chemicals (based on their MS/MS spectra) in >80% of the cases. This improved version called CFM-ID 3.0 is freely available as a web server. Its source code is also accessible online.
    Keywords:  MS spectral prediction; combinatorial fragmentation; liquid chromatography; mass spectrometry; metabolite identification; rule-based fragmentation; structure-based chemical classification
    DOI:  https://doi.org/10.3390/metabo9040072
  20. Sci Rep. 2019 Apr 23. 9(1): 6453
    Zhang P, Chan W, Ang IL, Wei R, Lam MMT, Lei KMK, Poon TCW.
      Fragmentation reactions of protonated α-amino acids (AAs) were studied previously using tandem mass spectrometry (MS/MS) of unit mass resolution. Isobaric fragmentation products and minor fragmentation products could have been overlooked or misannotated. In the present study, we examined the fragmentation patterns of 19 AAs using high-resolution electrospray ionization MS/MS (HR-ESI-MS/MS) with collision-induced dissociation (CID). Isobaric fragmentation products from protonated Met and Trp were resolved and identified for the first time. Previously unreported fragmentation products from protonated Met, Cys, Gln, Arg, and Lys were observed. Additionally, the chemical identity of a fragmentation product from protonated Trp that was incorrectly annotated in previous investigations was corrected. All previously unreported fragmentation products and reactions were verified by pseudo MS3 experiments and/or MS/MS analyses of deuterated AAs. Clearer pictures of the fragmentation reactions for Met, Cys, Trp, Gln, Arg and Lys were obtained in the present study.
    DOI:  https://doi.org/10.1038/s41598-019-42777-8
  21. Am J Clin Nutr. 2019 Apr 21. pii: nqz024. [Epub ahead of print]
    Woo SL, Yang J, Hsu M, Yang A, Zhang L, Lee RP, Gilbuena I, Thames G, Huang J, Rasmussen A, Carpenter CL, Henning SM, Heber D, Wang Y, Li Z.
      BACKGROUND: Recent studies have shown that circulating branched-chain amino acids (BCAAs) are elevated in obese, insulin-resistant individuals. However, it is not known if supplementation of additional BCAAs will further impair glucose metabolism.OBJECTIVES: The aim of this pilot study was to determine the effects of BCAA supplementation on glucose metabolism in obese, prediabetic individuals.
    METHODS: This is a randomized crossover study involving 12 obese individuals with prediabetes. Participants were randomly assigned to receive a daily supplement containing either 20 g BCAA or protein low in BCAAs for 4 wk with a 2-wk washout in between. At each visit, an oral-glucose-tolerance test (OGTT) was performed. Collected blood samples were used to measure glucose, insulin, and insulin resistance-associated biomarkers.
    RESULTS: BCAA supplementation tended to decrease the plasma glucose area under the curve (AUC) measured by the OGTT (AUC percentage change from supplementation baseline, BCAA: -3.3% ± 3%; low-BCAA: 10.0% ± 6%; P = 0.08). However, BCAA supplementation did not affect plasma insulin during OGTT challenge (BCAA: -3.9% ± 8%; low-BCAA: 14.8% ± 10%; P = 0.28). The plasma concentrations of nerve growth factor (BCAA: 4.0 ± 1 pg/mL; low-BCAA: 5.7 ± 1 pg/mL; P = 0.01) and monocyte chemoattractant protein-1 (BCAA: -0.4% ± 9%; low-BCAA: 29.0% ± 18%; P = 0.02) were significantly lowered by BCAA supplementation compared to low-BCAA control. Plasma interleukin 1β was significantly elevated by BCAA supplementation (BCAA: 231.4% ± 187%; low-BCAA: 20.6% ± 33%; P = 0.05). BCAA supplementation did not affect the circulating concentrations of the BCAAs leucine (BCAA: 9.0% ± 12%; low-BCAA: 9.2% ± 11%), valine (BCAA: 9.1% ± 11%; low-BCAA: 12.0% ± 13%), or isoleucine (BCAA: 2.5% ± 11%; low-BCAA: 7.3% ± 11%).
    CONCLUSIONS: Our data suggest that BCAA supplementation did not impair glucose metabolism in obese, prediabetic subjects. Further studies are needed to confirm the results seen in the present study. This study was registered at clinicaltrials.gov as NCT03715010.
    Keywords:  OGTT; branched-chain amino acid (BCAA); glucose metabolism; insulin resistance; obesity; prediabetes
    DOI:  https://doi.org/10.1093/ajcn/nqz024
  22. Proc Natl Acad Sci U S A. 2019 Apr 24. pii: 201814932. [Epub ahead of print]
    Arensman MD, Yang XS, Leahy DM, Toral-Barza L, Mileski M, Rosfjord EC, Wang F, Deng S, Myers JS, Abraham RT, Eng CH.
      T cell-invigorating cancer immunotherapies have near-curative potential. However, their clinical benefit is currently limited, as only a fraction of patients respond, suggesting that these regimens may benefit from combination with tumor-targeting treatments. As oncogenic progression is accompanied by alterations in metabolic pathways, tumors often become heavily reliant on antioxidant machinery and may be susceptible to increases in oxidative stress. The cystine-glutamate antiporter xCT is frequently overexpressed in cancer and fuels the production of the antioxidant glutathione; thus, tumors prone to redox stress may be selectively vulnerable to xCT disruption. However, systemic inhibition of xCT may compromise antitumor immunity, as xCT is implicated in supporting antigen-induced T cell proliferation. Therefore, we utilized immune-competent murine tumor models to investigate whether cancer cell expression of xCT was required for tumor growth in vivo and if deletion of host xCT impacted antitumor immune responses. Deletion of xCT in tumor cells led to defective cystine uptake, accumulation of reactive oxygen species, and impaired tumor growth, supporting a cancer cell-autonomous role for xCT. In contrast, we observed that, although T cell proliferation in culture was exquisitely dependent on xCT expression, xCT was dispensable for T cell proliferation in vivo and for the generation of primary and memory immune responses to tumors. These findings prompted the combination of tumor cell xCT deletion with the immunotherapeutic agent anti-CTLA-4, which dramatically increased the frequency and durability of antitumor responses. Together, these results identify a metabolic vulnerability specific to tumors and demonstrate that xCT disruption can expand the efficacy of anticancer immunotherapies.
    Keywords:  T cells; cancer; cystine; immunotherapy; xCT
    DOI:  https://doi.org/10.1073/pnas.1814932116
  23. Int J Mol Sci. 2019 Apr 25. pii: E2042. [Epub ahead of print]20(8):
    Kang H, Kim H, Lee S, Youn H, Youn B.
      Activation of epithelial-mesenchymal transition (EMT) is thought to be an essential step for cancer metastasis. Tumor cells undergo EMT in response to a diverse range of extra- and intracellular stimulants. Recently, it was reported that metabolic shifts control EMT progression and induce tumor aggressiveness. In this review, we summarize the involvement of altered glucose, lipid, and amino acid metabolic enzyme expression and the underlying molecular mechanisms in EMT induction in tumor cells. Moreover, we propose that metabolic regulation through gene-specific or pharmacological inhibition may suppress EMT and this treatment strategy may be applied to prevent tumor progression and improve anti-tumor therapeutic efficacy. This review presents evidence for the importance of metabolic changes in tumor progression and emphasizes the need for further studies to better understand tumor metabolism.
    Keywords:  EMT; cancer progression; metabolic reprogramming; metastasis
    DOI:  https://doi.org/10.3390/ijms20082042
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 08. pii: S1570-0232(19)30137-0. [Epub ahead of print]1117 136-147
    Gika H, Virgiliou C, Theodoridis G, Plumb RS, Wilson ID.
      Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these "routine" approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or "new" approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.
    Keywords:  Ion mobility spectrometry; Liquid chromatography; Mass spectrometry; Metabolomics; Metabonomics; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2019.04.009
  25. OMICS. 2019 Apr;23(4): 214-223
    Nalbantoglu S, Abu-Asab M, Suy S, Collins S, Amri H.
      Metabolomics offers new promise for research on prostate cancer (PCa) and its personalized treatment. Metabolomic profiling of radiation-treated PCa patients is particularly important to reveal their new metabolomic status, and evaluate the radiation effects. In addition, bioinformatics-integrated metabolomics-based approaches for disease profiling and assessment of therapy could help develop precision biomarkers in a context of PCa. We report mass spectrometry-based untargeted (global) serum metabolomics findings from patients with PCa (n = 55) before and after treatment with stereotactic body radiation therapy (SBRT), and intensity-modulated radiation therapy (IMRT) with SBRT, and using parsimony phylogenetic analysis. Importantly, the radiation-treated serum metabolome of patients represented a unique robust cluster on a cladogram that was distinct from the pre-RT metabolome. The altered radiation responsive serum metabolome was defined by predominant aberrations in the metabolic pathways of nitrogen, pyrimidine, purine, porphyrin, alanine, aspartate, glutamate, and glycerophospholipid. Our findings collectively suggest that global metabolomics integrated with parsimony phylogenetics offer a unique and robust systems biology analytical platform for powerful unbiased determination of radiotherapy (RT)-associated biosignatures in patients with PCa. These new observations call for future translational research for evaluation of metabolomic biomarkers in PCa prognosis specifically, and response to radiation treatment broadly. Radiation metabolomics is an emerging specialty of systems sciences and clinical medicine that warrants further research and educational initiatives.
    Keywords:  biomarkers and diagnostics; mass spectrometry; parsimony phylogenetics; prostate cancer; radiotherapy; untargeted (global) metabolomics
    DOI:  https://doi.org/10.1089/omi.2019.0006
  26. Methods Mol Biol. 2019 ;1987 51-64
    Dumit VI, Köttgen M, Hofherr A.
      Differential phosphorylation of proteins is a key regulatory mechanism in biology. Immunoprecipitation-coupled mass spectrometry facilitates the targeted analysis of transient receptor ion potential channel polycystin-2 (TRPP2) phosphorylation. However, empirical testing is required to optimize experimental conditions for immunoprecipitation and mass spectrometry. Here, we present a detailed workflow for the reliable analysis of endogenous TRPP2 phosphorylation in differentiated renal epithelial cells.
    Keywords:  ADPKD; Immunoprecipitation; Mass spectrometry; PKD2; Phosphoproteomics; Phosphorylation; Polycystic kidney disease; Post-translational modification; TRPP2
    DOI:  https://doi.org/10.1007/978-1-4939-9446-5_4
  27. J Sep Sci. 2019 Apr 23.
    Shi F, Wang C, Wang L, Song X, Yang H, Fu Q, Zhao W.
      Overcoming epidermal growth factor receptor resistance is a critical problem that needs to be solved in clinical practice. Drugs that downregulate the fatty acid synthase-epidermal growth factor receptor will become novel treatments for non-small cell lung cancer. Solanum nigrum, extracted with water at 4°C, shows strong cytotoxic activity and inhibits tumour growth in Lewis tumour bearing-mice in a dose-dependent manner. A novel active compound in Solanum nigrum, solaoiacid, was successfully separated and purified from Solanum nigrum by preparative HPLC-MS and ultra performance liquid chromatography time-of-flight-MS/MS. The IC50 of solaoiacid on lung cancer cells was 2.3 μmol/L, which was significantly lower than that of the known steroidal glycoalkaloid. Label-free proteomics and STRING Network analysis were used to identify significantly deregulated proteins in lung cancer cells that were treated with the fresh ripe fruit extracts of Solanum nigrum. Solanum nigrum regulates multiple signal pathways, including the epidermal growth factor receptor pathway. Solanum nigrum downregulated 24 main proteins with direct roles in fatty acid biosynthesis. Both Solanum nigrum and solaoiacid showed strong downregulation of the fatty acid synthase-epidermal growth factor receptor and anti-non-small cell lung cancer effects, and thus will become a novel drug for the treatment of non-small cell lung cancer. This article is protected by copyright. All rights reserved.
    Keywords:  Solanum nigrum berries; epidermal growth factor receptor; fatty acid synthase; non-small cell lung cancer; proteomics
    DOI:  https://doi.org/10.1002/jssc.201801165
  28. Inorg Chem. 2019 Apr 23.
    Jin S, Guo Y, Song D, Zhu Z, Zhang Z, Sun Y, Yang T, Guo Z, Wang X.
      Cancer is characterized by abnormal cellular energy metabolism, which preferentially switches to aerobic glycolysis rather than oxidative phosphorylation as a means of glucose metabolism. Many key enzymes involved in the abnormal glycolysis are potential targets of anticancer drugs. Platinum(IV) complexes are potential anticancer prodrugs and kinetically more inert than the platinum(II) counterparts, which offer an opportunity to be modified by functional ligands for activation or targeted delivery. A novel platinum(IV) complex, c, c, t-[Pt(NH3)2Cl2(C10H15N2O3S)(C2HO2Cl2)] (DPB), was designed to explore the effects of axial ligands on the reactivity and bioactivity of the complex as well as on tumor energy metabolism. The complex was characterized by electrospray ionization mass spectrometry and multinuclear (1H, 13C, and 195Pt) NMR spectroscopy. The introduction of dichloroacetate (DCA) markedly increases the lipophilicity, reactivity, and cytotoxicity of the complex and blocks the growth of cancer cells having active glycolysis, and the introduction of biotin (C10H16N2O3S) enhances the tumor-targeting potential of the complex. The cytotoxicity of DPB is increased dramatically in a variety of cancer cell lines as compared with the platinum(IV) complex PB without the DCA group. DPB alters the mitochondrial membrane potential and disrupts the mitochondrial morphology. The levels of mitochondrial and cellular reactive oxygen species are also decreased. Furthermore, the mitochondrial function of tumor cells was impaired by DPB, leading to the inhibition of both glycolysis and glucose oxidation and finally to the death of cancer cells via a mitochondria-mediated apoptotic pathway. These findings demonstrate that DPB suppresses cancer cells mainly through altering metabolic pathways and highlight the importance of dual-targeting for the efficacy of anticancer drugs.
    DOI:  https://doi.org/10.1021/acs.inorgchem.9b00708
  29. Toxicol Appl Pharmacol. 2019 Apr 19. pii: S0041-008X(19)30146-2. [Epub ahead of print]
    Cui Y, Han J, Ren J, Chen H, Xu B, Song N, Li H, Liang A, Shen G.
      As the main toxic component of aristolochic acid, aristolochic acid I (AAI) is primarily found in Aristolochiaceae plants such as Aristolochia, Aristolochia fangchi and Caulis aristolochiae manshuriensis. AAI has been proven to be carcinogenic, mutagenic and nephrotoxic. Although the role of AAI in testicular toxicity has been reported, its mechanism of action is unknown. Using metabonomics and molecular biology techniques, we tried to identify the differential endogenous metabolites of AAI that may affect the changes in testicular function in mice, map the network of metabolic pathways, and systematically reveal the molecular mechanism of AAI-induced testicular toxicity. We found that AAI inhibited amino acid metabolism in mouse testicular cells, impeded the uptake and oxidative decomposition of fatty acids, prevented normal glucose uptake by testicular cells, which inhibited glycolysis and gluconeogenesis, affected the mitochondrial tricarboxylic acid (TCA) cycle, which impaired the ATP energy supply, decreased the number of spermatogenic cells and sperm in the testes, induced changes in the mitochondrial state of spermatogonial cells, and ultimately led to physiological and pathological changes in the testes. AAI also regulated the testicular physiological activity by regulating the androgen receptor and hormone levels. This study used metabonomics and other methods to elucidate the mechanism of AAI-induced testicular toxicity from a new angle.
    Keywords:  ATP; Aristolochic acid I; Metabonomics; TCA cycle
    DOI:  https://doi.org/10.1016/j.taap.2019.04.014
  30. Anal Bioanal Chem. 2019 Apr 23.
    Szykuła KM, Meurs J, Turner MA, Creaser CS, Reynolds JC.
      Untargeted metabolite profiling of biological samples is a challenge for analytical science due to the high degree of complexity of biofluids. Isobaric species may also not be resolved using mass spectrometry alone. As a result of these factors, many potential biomarkers may not be detected or are masked by co-eluting interferences in conventional LC-MS metabolomic analyses. In this study, a comprehensive liquid chromatography-mass spectrometry workflow incorporating a fast-scanning miniaturised high-field asymmetric waveform ion mobility spectrometry separation (LC-FAIMS-MS) is applied to the untargeted metabolomic analysis of human urine. The time-of-flight mass spectrometer used in the study was scanned at a rate of 20 scans s-1 enabling a FAIMS CF spectrum to be acquired within a 1-s scan time, maintaining an adequate number of data points across each LC peak. The developed method is demonstrated to be able to resolve co-eluting isomeric species and shows good reproducibility (%RSD < 4.9%). The nested datasets obtained for fresh, aged, and QC urine samples were submitted for multivariate statistical analysis. Seventy unique biomarker ions showing a statistically significant difference between fresh and aged urine were identified with optimal transmission CF values obtained across the full CF spectrum. The potential of using FAIMS to select ions for in-source collision-induced dissociation is demonstrated for FAIMS-selected methylxanthine ions yielding characteristic fragment ion species indicative of the precursor. Graphical abstract.
    Keywords:  Bioanalytical methods; Differential mobility; Ion mobility; Mass spectrometry; Metabolomics; Separations
    DOI:  https://doi.org/10.1007/s00216-019-01790-6
  31. Physiol Rep. 2019 Apr;7(8): e14040
    Gollasch B, Dogan I, Rothe M, Gollasch M, Luft FC.
      Omega-3 fatty acids have long been ascribed a positive cardiovascular function. However, the plasma measurements invariably ignore 40% of the blood specimen, cells that engage in continuous exchange with their environment. In our study, we included all components of the circulating blood. Erythrocyte or red-blood-cell (RBC) n-3 fatty acid status has been linked to cardiovascular disease and death. A low omega-3 index is an independent risk factor for cardiovascular disease and mortality. We tested the hypothesis that acute, maximal exercise would influence the relationship between RBC and serum fatty acids. RBC fatty acids profiling was achieved using targeted HPLC-MS mass spectrometry. Healthy volunteers performed maximal treadmill exercise testing using the modified Bruce protocol. Central hemodynamics were monitored and maximal workload was assessed in metabolic equivalents (METs). Venous blood was obtained for RBC lipidomics. With the incremental exercise test, no fatty acid-level variations were found in RBCs, while heart rate and arterial blood pressure increased significantly. No changes occurred in the omega-3 quotient, namely the percentage of eicosapentaenoic acid and docosahexaenoic acid in RBC fatty acids in the RBC membrane. Nonetheless, maximal (13.50 ± 1.97 METs) exercise intensity led to a decrease of RBC lauric acid (C12:0) in the recovery period. These data suggest that despite significant hemodynamic effects, short-term maximal exercise is insufficient to alter RBC n-3 and other fatty-acid status, including the omega-3 quotient, in healthy individuals. RBC lauric acid deserves further scrutiny as a potential regulator of cardiovascular and metabolic functions.
    Keywords:  Erythrocytes; exercise; fatty acids; lipidomics
    DOI:  https://doi.org/10.14814/phy2.14040
  32. Biochem Biophys Res Commun. 2019 Apr 20. pii: S0006-291X(19)30743-0. [Epub ahead of print]
    Huang Z, Xia L, Zhou X, Wei C, Mo Q.
      Arachidonate lipoxygenase12 (Alox12) and its metabolites 12S-hydroxyeicosatetraenoic acid (12S-HETE) have been implicated in influencing tumor transformation and progression. In this study, we have systematically evaluated the expression, function and the downstream effectors of Alox12 in breast cancer using loss- and gain-of-function approaches. We demonstrated that both mRNA and protein levels of Alox12 were significantly increased in multiple breast cancer cell lines compared to normal breast cells. The upregulation of Alox12 expression was also observed in breast cancer tissues and their matched normal breast tissues obtained from patients. Functionally, we demonstrated that Alox12 overexpression was sufficient to stimulate growth in normal breast cells but not breast cancer cells. This also protects breast cancer cell from chemotherapy-induced growth arrest and apoptosis. In contrast, Alox12 depletion inhibited breast cancer growth and survival, and significantly enhanced the chemotherapeutic agents' efficacy. Mechanism studies showed that Alox12 depletion activated AMP-activated protein kinase (AMPK), leading to the inhibition of acetyl-CoA carboxylase1 (ACC1) enzyme activity and lipid synthesis. The recuse of the effects of Alox12 depletion using Alox12 metabolites 12S-HETE further confirmed that AMPK and its subsequent inhibition of ACC1 activity and lipid synthesis were the downstream signaling of Alox12 inhibition. Our findings highlighted the important role of Alox12 in breast cancer, particularly in response to chemotherapy. Our work also demonstrate that inhibiting Alox12 is a possible alternative therapeutic strategy to overcome chemoresistance in breast cancer.
    Keywords:  12(S)-HETE; AMPK; Alox12; Breast cancer; Chemoresistance; Lipid synthesis
    DOI:  https://doi.org/10.1016/j.bbrc.2019.04.101
  33. J Cell Biochem. 2019 Apr 24.
    Xu M, Jiang F, Li B, Zhang Z.
      Lipid accumulation is a vital event in the progression of diabetic nephropathy. 1,25-Dihydroxyvitamin D3 (1α,25(OH)2 D3 ) is considered to have a protective effect on diabetic nephropathy. However, it remains unclear whether 1α,25(OH)2 D3 can inhibit lipid accumulation, and the potential mechanisms responsible for lipid metabolism are incompletely understood. In this study, we evaluated the effects of 1α,25(OH)2 D3 on lipid metabolism in high glucose-exposed rat renal tubular epithelial NRK-52E cells. Results indicated that high glucose-enhanced lipid accumulation in NRK-52E cells and 1α,25(OH)2 D3 can remarkably decrease high glucose-induced lipid accumulation. Western blot showed that 1α,25(OH)2 D3 alleviated high glucose-induced upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and SREBP2, along with their established target genes fatty acid synthase (FASN) and hydroxymethylglutaryl CoA reductases (HMGCR). Overall, these findings suggest that 1α,25(OH)2 D3 downregulated the expressions of SREBPs to inhibit high glucose-induced lipid accumulation, which provides new sights into the protective effects of 1α,25(OH)2 D3 on diabetic nephropathy.
    Keywords:  1,25-dihydroxyvitamin D3; cholesterol; feedback response; high glucose; lipid droplets
    DOI:  https://doi.org/10.1002/jcb.28786
  34. Cell Mol Gastroenterol Hepatol. 2019 Apr 17. pii: S2352-345X(19)30046-3. [Epub ahead of print]
    Esler WP, Bence KK.
      The prevalence and diagnosis of nonalcoholic fatty liver disease (NAFLD) is on the rise worldwide and currently has no FDA-approved pharmacotherapy. The increase in disease burden of NAFLD and a more severe form of this progressive liver disease, nonalcoholic steatohepatitis (NASH), largely mirrors the increase in obesity and type 2 diabetes (T2D) and reflects the hepatic manifestation of an altered metabolic state. Indeed, metabolic syndrome, defined as a constellation of obesity, insulin resistance, hyperglycemia, dyslipidemia and hypertension, is the major risk factor predisposing the NAFLD and NASH. There are multiple potential pharmacologic strategies to rebalance aspects of disordered metabolism in NAFLD. These include therapies aimed at reducing hepatic steatosis by directly modulating lipid metabolism within the liver, inhibiting fructose metabolism, altering delivery of free fatty acids from the adipose to the liver by targeting insulin resistance and/or adipose metabolism, modulating glycemia, and altering pleiotropic metabolic pathways simultaneously. Emerging data from human genetics also supports a role for metabolic drivers in NAFLD and risk for progression to NASH. In this review, we highlight the prominent metabolic drivers of NAFLD pathogenesis and discuss the major metabolic targets of NASH pharmacotherapy.
    DOI:  https://doi.org/10.1016/j.jcmgh.2019.04.007
  35. Cancer Discov. 2019 Apr 23. pii: CD-18-1218. [Epub ahead of print]
    Pantelidou C, Sonzogni O, de Oliveira Taveira M, Mehta AK, Kothari A, Wang D, Visal T, Li MK, Pinto J, Castrillon JA, Cheney EM, Bouwman P, Jonkers J, Rottenberg S, Guerriero JL, Wulf GM, Shapiro GI.
      Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T cell infiltration and activation in vivo, and that CD8+ T cell depletion severely compromises anti-tumor efficacy. Olaparib-induced T cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared to HR-proficient TNBC cells and in vivo models. CRISPR-knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC.
    DOI:  https://doi.org/10.1158/2159-8290.CD-18-1218
  36. Metabolites. 2019 Apr 13. pii: E71. [Epub ahead of print]9(4):
    Benedetti S, Al-Tannak NF, Alzharani M, Moir HJ, Stensel DJ, Thackray AE, Naughton DP, Dorak MT, Spendiff O, Hill N, Watson DG, Allgrove J.
      South Asians have a greater cardiovascular disease (CVD) and type 2 diabetes (T2D) risk than white Europeans, but the mechanisms are poorly understood. This study examined ethnic differences in free fatty acids (FFAs) metabolic profile (assessed using liquid chromatography-mass spectrometry), appetite-related hormones and traditional CVD and T2D risk markers in blood samples collected from 16 South Asian and 16 white European men and explored associations with body composition, objectively-measured physical activity and cardiorespiratory fitness. South Asians exhibited higher concentrations of five FFAs (laurate, myristate, palmitate, linolenic, linoleate; p ≤ 0.040), lower acylated ghrelin (ES = 1.00, p = 0.008) and higher leptin (ES = 1.11, p = 0.004) than white Europeans; total peptide YY was similar between groups (p = 0.381). South Asians exhibited elevated fasting insulin, C-reactive protein, interleukin-6, triacylglycerol and ratio of total cholesterol to high-density lipoprotein cholesterol (HDL-C) and lower fasting HDL-C (all ES ≥ 0.74, p ≤ 0.053). Controlling for body fat percentage (assessed using air displacement plethysmography) attenuated these differences. Despite similar habitual moderate-to-vigorous physical activity (ES = 0.18, p = 0.675), V ˙ O2max was lower in South Asians (ES = 1.36, p = 0.001). Circulating FFAs in South Asians were positively correlated with body fat percentage (r2 = 0.92), body mass (r2 = 0.86) and AUC glucose (r2 = 0.89) whereas in white Europeans FFAs were negatively correlated with total step counts (r2 = 0.96). In conclusion, South Asians exhibited a different FFA profile, lower ghrelin, higher leptin, impaired CVD and T2D risk markers and lower cardiorespiratory fitness than white Europeans.
    Keywords:  South Asian; Type 2 diabetes; appetite hormones; cardiorespiratory fitness; cardiovascular disease; free fatty acids; inflammation; metabolic markers; metabolomics; physical activity
    DOI:  https://doi.org/10.3390/metabo9040071
  37. Electrophoresis. 2019 Apr 26.
    Zhang W, Segers K, Mangelings D, Van Eeckhaut A, Hankemeier T, Heyden YV, Ramautar R.
      In metabolomics, CE-MS is a useful analytical technique for the profiling of polar and charged metabolites. However, the actual utility of this approach for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by conventional CE-MS at low-pH separation conditions was performed, using pooled human plasma samples with spiked biomarkers. In this context, two studies have been carried out in this work. The focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope-labeled compounds, whereas another set (class II) was spiked with six different isotope-labeled compounds. In study II, the focus was also on comparing two sets of plasma samples, however, in this case the isotope-labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes, and with the absence of one compound in each class. In both studies, blank pooled human plasma was used as a quality control sample. The main aim was to determine whether the CE-MS-based metabolomics approach could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (i.e., MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope-labeled compounds, thereby emphasizing the utility of CE-MS in metabolomics for biomarker discovery. This article is protected by copyright. All rights reserved.
    Keywords:  Mass spectrometry; Metabolic profiling; Metabolomics; Spiked biomarkers; Validation
    DOI:  https://doi.org/10.1002/elps.201900126
  38. Clin Proteomics. 2019 ;16 16
    Dey KK, Wang H, Niu M, Bai B, Wang X, Li Y, Cho JH, Tan H, Mishra A, High AA, Chen PC, Wu Z, Beach TG, Peng J.
      Background: Blood-based protein measurement is a routine practice for detecting biomarkers in human disease. Comprehensive profiling of blood/plasma/serum proteome is a challenge due to an extremely large dynamic range, as exemplified by a small subset of highly abundant proteins. Antibody-based depletion of these abundant proteins alleviates the problem but introduces experimental variations. We aimed to establish a method for direct profiling of undepleted human serum and apply the method toward biomarker discovery for Alzheimer's disease (AD), as AD is the most common form of dementia without available blood-based biomarkers in clinic.Methods: We present an ultra-deep analysis of undepleted human serum proteome by combining the latest 11-plex tandem-mass-tag (TMT) labeling, exhaustive two-dimensional liquid chromatography (LC/LC) fractionation (the 1st LC: 3 h for 180 fractions, and the 2nd LC: 3 h gradient per fraction), coupled with high resolution tandem mass spectrometry (MS/MS). AD (n = 6) and control (n = 5) sera were analyzed in this pilot study. In addition, we implemented a multiplexed targeted LC-MS3 method (TOMAHAQ) for the validation of selected target proteins.
    Results: The TMT-LC/LC-MS/MS platform is capable of analyzing 4826 protein components (4368 genes), covering at least 6 orders of magnitude in dynamic range, representing one of the deepest serum proteome analysis. We defined intra- and inter- group variability in the AD and control groups. Statistical analysis revealed differentially expressed proteins in AD (26 decreased and 4 increased). Notably, these altered proteins are enriched in the known pathways of mitochondria, fatty acid beta oxidation, and AGE/RAGE. Finally, we set up a TOMAHAQ method to confirm the decrease of PCK2 and AK2 in our AD samples.
    Conclusions: Our results show an ultra-deep serum discovery study by TMT-LC/LC-MS/MS, and a validation experiment by TOMAHAQ targeted LC-MS3. The MS-based discovery and validation methods are of general use for biomarker discovery from complex biofluids (e.g. serum proteome). This pilot study also identified deregulated proteins, in particular proteins associated with mitochondrial function in the AD serum samples. These proteins may serve as novel AD candidate biomarkers.
    Keywords:  Alzheimer’s disease; Biomarker; Human blood; Mass spectrometry; Plasma; Proteome; Proteomics; Serum; Tandem mass tag
    DOI:  https://doi.org/10.1186/s12014-019-9237-1
  39. Molecules. 2019 Apr 25. pii: E1639. [Epub ahead of print]24(8):
    Liakh I, Pakiet A, Sledzinski T, Mika A.
      Oxylipins are potent lipid mediators derived from polyunsaturated fatty acids, which play important roles in various biological processes. Being important regulators and/or markers of a wide range of normal and pathological processes, oxylipins are becoming a popular subject of research; however, the low stability and often very low concentration of oxylipins in samples are a significant challenge for authors and continuous improvement is required in both the extraction and analysis techniques. In recent years, the study of oxylipins has been directly related to the development of new technological platforms based on mass spectrometry (LC-MS/MS and gas chromatography-mass spectrometry (GC-MS)/MS), as well as the improvement in methods for the extraction of oxylipins from biological samples. In this review, we systematize and compare information on sample preparation procedures, including solid-phase extraction, liquid-liquid extraction from different biological tissues.
    Keywords:  biological samples; liquid–liquid extraction; oxylipins; protein precipitation; sample preparation; solid-phase extraction
    DOI:  https://doi.org/10.3390/molecules24081639
  40. J Extracell Vesicles. 2019 ;8(1): 1596669
    Palviainen M, Saari H, Kärkkäinen O, Pekkinen J, Auriola S, Yliperttula M, Puhka M, Hanhineva K, Siljander PR.
      One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.
    Keywords:  Conditioned medium; bioreactor; cell culture; extracellular vesicles (EVs); metabolomics; prostate cancer cell line
    DOI:  https://doi.org/10.1080/20013078.2019.1596669
  41. J Endocrinol Invest. 2019 Apr 22.
    Bianchi L, Campi B, Sessa MR, De Marco G, Ferrarini E, Zucchi R, Marcocci C, Vitti P, Manetti L, Saba A, Agretti P.
      PURPOSE: One of the best indicators of adrenal gland dysfunction is the level of free cortisol measured in the 24-h urine (UFC) which faithfully reflects the level of biologically active serum cortisol not subjected to circadian variations. Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a sensitive, accurate and precise method recently available in routine laboratories that could remedy interference problems of immunoassays.METHODS: In this study, a literature reference range for UFC measured by LC-MS-MS was verified, and UFC values measured by LC-MS-MS and immunoassay were compared. Immunometric UFC measurement was performed by ACCESS CORTISOL assay without preliminary extraction, using Beckman Coulter UniCel DxI 600 highly automated platform. Liquid chromatography-tandem mass spectrometry UFC measurement was performed by a home-made validated method using cortisol-D4 as internal standard with preliminary deproteinization of urinary samples by centrifugal filter and injection on reverse-phase column. Cortisol was analyzed in positive ion mode with an ESI interface.
    RESULTS: The reference interval from literature (11-70 μg/day) was confirmed by results obtained for healthy study group. Comparison study of the two methods highlighted a constant and proportional systematic error with a general tendency to overestimate results for the in-use method.
    CONCLUSIONS: In conclusion, the direct immunometric method overestimates UFC results with respect to liquid chromatography-tandem mass spectrometry which represents the reference method. The literature reference range 11-70 μg/day was confirmed and can be adopted by our lab that will shift all UFC tests performed in routine to the mass spectrometry-based method, satisfying clinicians' request.
    Keywords:  Antibody cross-reactivity; Immunometric assay; Interference from steroid similar molecules; LC–MS–MS; Reference range; Urinary free cortisol
    DOI:  https://doi.org/10.1007/s40618-019-01050-5
  42. Eur J Pharmacol. 2019 Apr 20. pii: S0014-2999(19)30266-3. [Epub ahead of print]
    Phang CW, Gandah NA, Abd Malek SN, Karsani SA.
      Flavokawain C (FKC), a naturally occurring chalcone, has previously been shown to inhibit the growth of colon carcinoma HCT 116 cells through induction of apoptosis and cell cycle arrest. However, the possible underlying mechanisms of cell death as a response to FKC treatment remains unclear. In this study, we performed proteomic analysis of HCT 116 cells treated with FKC to identify proteins that change in abundance. This was followed by bioinformatic analysis to predict possible associated molecular targets or pathways involved in the observed effects of FKC. A total of 35 proteins that changed in abundance (17 increased and 18 decreased) were identified through two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Using the Ingenuity Pathway Analysis (IPA), these proteins were predicted to be involved in cell death and survival, cell cycle, cellular growth and proliferation, protein synthesis, post-translational modification and amino acid metabolism by. Further analysis of the transcript levels of selected proteins using qPCR showed that some of the genes exhibited similar change of profile to that of the proteins'. Our results have provided novel insights into the potential molecular mechanisms underlying FKC-induced apoptosis or cell death in colon cancer cells.
    Keywords:  Chalcone; Colon cancer cells; Flavokawain C; Potential anti-cancer; Proteomics
    DOI:  https://doi.org/10.1016/j.ejphar.2019.04.032
  43. Angew Chem Int Ed Engl. 2019 Apr 23.
    Mu C, Wang J, Barraza KM, Zhang X, Beauchamp JL.
      Even though the general mechanism of photodynamic therapy for cancer is known, the details and consequences of the reactions between the photosensitizer generated singlet oxygen and substrate molecules remain elusive at the molecular level. Using temoporfin as the photosensitizer, here we combine field-induced droplet ionization mass spectrometry and acoustic levitation techniques to study the "wall-less" oxidation reactions of 18:1 cardiolipin and POPG mediated by singlet oxygen at the air-water interface of levitated water droplets. For both cardiolipin and POPG, every unsaturated oleyl chain is oxidized into an allyl hydroperoxide, which surprisingly is immune to further oxidation. This is attributed to the increased hydrophilicity of the oxidized chain, attracting it toward the water phase, increasing membrane permeability and eventually triggering cell death.
    Keywords:  Acoustic levitation; Air-water interface; Lipid oxidation; Mass spectrometry; photodynamic therapy
    DOI:  https://doi.org/10.1002/anie.201902815
  44. Free Radic Biol Med. 2019 Apr 19. pii: S0891-5849(18)32425-0. [Epub ahead of print]
    Giusepponi D, Galarini R, Barola C, Torquato P, Bartolini D, Moretti S, Saluti G, Gioiello A, Libetta C, Galli F.
      The role of vitamin E in both enzymatic and free radical-dependent metabolism of polyunsaturated fatty acids (PUFAs) has been well demonstrated. This study proposed a new LC-MS/MS method to quantify the main vitamin E forms, their metabolites and main PUFA species in human blood, since, at present, there are not procedures able to simultaneously determine these two classes of compounds. After the optimization of sample treatment and reverse-phase separation conditions, tandem mass spectrometry detection was evaluated experimenting both positive and negative electrospray ionization modes. The procedure was also preliminarily adapted to assess five arachidonic acid-derived eicosanoids that could be under the influence of vitamin E function, such as LTB4 (leukotriene B4), 20-HETE (20-hydroxyeicosatetraenoic acid) and their ω-oxidation metabolites. After the validation study, the performance characteristics were confirmed analysing a certified reference material (SRM® 1950 - frozen human plasma by NIST). Finally, an application of the method in the analysis of lipid abnormalities of chronic kidney disease patients was shown.
    Keywords:  Eicosanoids; Human plasma/serum; Liquid-chromatography tandem-mass spectrometry (LC-MS/MS); Polyunsaturated fatty acids; Tocopheryl quinone; Validation; Vitamin E
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.04.017
  45. Front Mol Biosci. 2019 ;6 22
    Han Q, Phillips RS, Li J.
      
    Keywords:  aromatic amino acids; auxin; dopamine; kynurenine; melatonin; metabolism; serotoinin; tryptophan
    DOI:  https://doi.org/10.3389/fmolb.2019.00022
  46. Nat Rev Cancer. 2019 Apr 25.
    Srivastava S, Koay EJ, Borowsky AD, De Marzo AM, Ghosh S, Wagner PD, Kramer BS.
      For cancer screening to be successful, it should primarily detect cancers with lethal potential or their precursors early, leading to therapy that reduces mortality and morbidity. Screening programmes have been successful for colon and cervical cancers, where subsequent surgical removal of precursor lesions has resulted in a reduction in cancer incidence and mortality. However, many types of cancer exhibit a range of heterogeneous behaviours and variable likelihoods of progression and death. Consequently, screening for some cancers may have minimal impact on mortality and may do more harm than good. Since the implementation of screening tests for certain cancers (for example, breast and prostate cancers), a spike in incidence of in situ and early-stage cancers has been observed, but a link to reduction in cancer-specific mortality has not been as clear. It is difficult to determine how many of these mortality reductions are due to screening and how many are due to improved treatments of tumours. In cancers with lower incidence but high mortality (for example, pancreatic cancer), screening has focused on high-risk populations, but challenges similar to those for general population screening remain, particularly with regard to finding lesions with difficult-to-characterize malignant potential (for example, intraductal papillary mucinous neoplasms). More sensitive screening methods are detecting smaller and smaller lesions, but this has not been accompanied by a comparable reduction in the incidence of invasive cancers. In this Opinion article, we focus on the contribution of screening in general and high-risk populations to overdiagnosis, the effects of overdiagnosis on patients and emerging strategies to reduce overdiagnosis of indolent cancers through an understanding of tumour heterogeneity, the biology of how cancers evolve and progress, the molecular and cellular features of early neoplasia and the dynamics of the interactions of early lesions with their surrounding tissue microenvironment.
    DOI:  https://doi.org/10.1038/s41568-019-0142-8
  47. EBioMedicine. 2019 Apr 19. pii: S2352-3964(19)30270-1. [Epub ahead of print]
    Qi D, Wu E.
      
    Keywords:  CRC; Prognosis; TME; TNM
    DOI:  https://doi.org/10.1016/j.ebiom.2019.04.031
  48. Nat Commun. 2019 Apr 23. 10(1): 1832
    Dadsena S, Bockelmann S, Mina JGM, Hassan DG, Korneev S, Razzera G, Jahn H, Niekamp P, Müller D, Schneider M, Tafesse FG, Marrink SJ, Melo MN, Holthuis JCM.
      Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.
    DOI:  https://doi.org/10.1038/s41467-019-09654-4
  49. Pediatr Int. 2019 Apr;61(4): 415-416
    Suganuma H, McPhee AJ, Bratkovic D, Gibson RA, Andersen CC.
      
    Keywords:  arachidonic acid; docosahexaenoic acid; long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; preterm infant
    DOI:  https://doi.org/10.1111/ped.13802