bims-mascan 2022-05-15 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2022–05–15
twenty-two papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.
Editorial: Metabolite and Nutrient Transporters in Cancer-Cell Metabolism: Role in Cancer Progression and Metastasis.
Building Spectral Libraries from Narrow-Window Data-Independent Acquisition Mass Spectrometry Data.
Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.
Role of Amino Acid Transporter SNAT1/SLC38A1 in Human Melanoma.
SCD1/FADS2 fatty acid desaturases equipoise lipid metabolic activity and redox-driven ferroptosis in ascites-derived ovarian cancer cells.
A multimodal analytical method to simultaneously determine monoacetyldiacylglycerols, medium and long chain triglycerides in biological samples during routine lipidomics.
Precise Metabolomics Reveals a Diversity of Aging-Associated Metabolic Features.
Single-cell mass spectrometry.
Improving confidence in lipidomic annotations by incorporating empirical ion mobility regression analysis and chemical class prediction.
Application of Metabolite Set Enrichment Analysis (MSEA) on untargeted metabolomics data prioritizes relevant pathways and detects novel biomarkers for inherited metabolic disorders.
Mass defect filter technique combined with stable isotope tracing for drug metabolite identification using high-resolution mass spectrometry.
Metabolic outliers in human disease.
Lipid Metabolism and Ferroptosis.
Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.
Lipin Expression Correlates to Differences in Phospholipid Profiles in Drug-Resistant Prostate Cancer Cells.
Mass Spectrometry Based Subcellular Coenzyme Analysis.
The Role of Extracellular Vesicles in Metabolic Reprogramming of the Tumor Microenvironment.
Exploring the Role of Metabolites in Cancer and the Associated Nerve Crosstalk.
An Organ-Specific Metabolite Annotation Approach for Ambient Mass Spectrometry Imaging Reveals Spatial Metabolic Alterations of a Whole Mouse Body.
Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration.
Hypoxia-reoxygenation couples 3βHSD1 enzyme and cofactor upregulation to facilitate androgen biosynthesis and hormone therapy resistance in prostate cancer.

J Exp Bot. 2022 May 13. 73(9): 2938-2952

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.

Somnath Koley, Kevin L Chu, Saba S Gill, Doug K Allen.

  Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.

Keywords:  Central metabolism; ion suppression and enhancement; isomer separation; isotopic labeling; liquid chromatography–mass spectrometry; metabolite quantification; mixed-mode column chromatography; oilseeds

DOI:  https://doi.org/10.1093/jxb/erac062
Front Cell Dev Biol. 2022 ;10 885717

Editorial: Metabolite and Nutrient Transporters in Cancer-Cell Metabolism: Role in Cancer Progression and Metastasis.

Vadivel Ganapathy, Sebastian Haferkamp, Eric K Parkinson, Maria E Mycielska.



Keywords:  Warburg effect; amino acids; cancer-cell metabolism; carnitine; citrate; glucose; metabolomics; nutrient/acid/base transporters

DOI:  https://doi.org/10.3389/fcell.2022.885717
J Proteome Res. 2022 May 12.

Building Spectral Libraries from Narrow-Window Data-Independent Acquisition Mass Spectrometry Data.

Lilian R Heil, William E Fondrie, Christopher D McGann, Alexander J Federation, William S Noble, Michael J MacCoss, Uri Keich.

  Advances in library-based methods for peptide detection from data-independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high-quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico, but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate-controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for accurate peptide detection and quantitation in wide-window DIA data. We compare the results of this search workflow to other library-free approaches and demonstrate that our search is competitive in terms of accuracy and sensitivity. These results demonstrate that the proposed workflow has the capacity to generate spectral libraries while avoiding the limitations of other methods.

Keywords:  data-independent acquisition; database search; spectral library

DOI:  https://doi.org/10.1021/acs.jproteome.1c00895
J Lipid Res. 2022 May 07. pii: S0022-2275(22)00056-6. [Epub ahead of print] 100223

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

Reuben S E Young, Andrew P Bowman, Kaylyn D Tousignant, Berwyck L J Poad, Jennifer H Gunter, Lisa K Philp, Colleen C Nelson, Shane R Ellis, Ron M A Heeren, Martin C Sadowski, Stephen J Blanksby.

  The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labelled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome, and induced repartitioning of n-3 and n-6 polyunsaturated FAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Keywords:  Fatty acid/Transport; Imaging Mass Spectrometry; Lipase; Lipid Isomers; Lipolysis and fatty acid metabolism; Ozone-Induced Dissociation; Phospholipid/Metabolism; Phospholipids/Phosphatidylcholine; Stable-Isotope Tracing

DOI:  https://doi.org/10.1016/j.jlr.2022.100223
Cancers (Basel). 2022 Apr 26. pii: 2151. [Epub ahead of print]14(9):

Role of Amino Acid Transporter SNAT1/SLC38A1 in Human Melanoma.

Ines Böhme-Schäfer, Sandra Lörentz, Anja Katrin Bosserhoff.

  The tumor metabolism is an important driver of cancer cell survival and growth, as rapidly dividing tumor cells exhibit a high demand for energetic sources and must adapt to microenvironmental changes. Therefore, metabolic reprogramming of cancer cells and the associated deregulation of nutrient transporters are a hallmark of cancer cells. Amino acids are essential for cancer cells to synthesize the necessary amount of protein, DNA, and RNA. Although cancer cells can synthesize glutamine de novo, most cancer cells show an increased uptake of glutamine from the tumor microenvironment. Especially SNAT1/SLC38A1, a member of the sodium neutral amino acid transporter (SNAT) family, plays an essential role during major net import of glutamine. In this study, we revealed a significant upregulation of SNAT1 expression in human melanoma tissue in comparison to healthy epidermis and an increased SNAT1 expression level in human melanoma cell lines when compared to normal human melanocytes (NHEMs). We demonstrated that functional inhibition of SNAT1 with α-(methylamino) isobutyric acid (MeAIB), as well as siRNA-mediated downregulation reduces cancer cell growth, cellular migration, invasion, and leads to induction of senescence in melanoma cells. Consequently, these results demonstrate that the amino acid transporter SNAT1 is essential for cancer growth, and indicates a potential target for cancer chemotherapy.

Keywords:  amino acid transporter; melanoma; tumor metabolism

DOI:  https://doi.org/10.3390/cancers14092151
Theranostics. 2022 ;12(7): 3534-3552

SCD1/FADS2 fatty acid desaturases equipoise lipid metabolic activity and redox-driven ferroptosis in ascites-derived ovarian cancer cells.

Yang Xuan, Huogang Wang, Mingo Mh Yung, Fushun Chen, Wai-Sun Chan, Yau-Sang Chan, Stephen Kw Tsui, Hextan Ys Ngan, Karen Kl Chan, David W Chan.

  Rationale: Malignant ascites in peritoneal metastases is a lipid-enriched microenvironment and is frequently involved in the poor prognosis of epithelial ovarian cancer (EOC). However, the detailed mechanisms underlying ovarian cancer (OvCa) cells dictating their lipid metabolic activities in promoting tumor progression remain elusive. Methods: The omental conditioned medium (OCM) was established to imitate the omental or ascites microenvironment. Mass spectrometry, RT-qPCR, IHC, and western blot assays were applied to evaluate human fatty acid desaturases expressions and activities. Pharmaceutical inhibition and genetic ablation of SCD1/FADS2 were performed to observe the oncogenic capacities. RNA sequencing, lipid peroxidation, cellular iron, ROS, and Mito-Stress assays were applied to examine ferroptosis. OvCa patient-derived organoid and mouse model of peritoneal metastases were used to evaluate the combined effect of SCD1/FADS2 inhibitors with cisplatin. Results: We found that two critical fatty acid desaturases, stearoyl-CoA desaturase-1 (SCD1) and acyl-CoA 6-desaturase (FADS2), were aberrantly upregulated, accelerating lipid metabolic activities and tumor aggressiveness of ascites-derived OvCa cells. Lipidomic analysis revealed that the elevation of unsaturated fatty acids (UFAs) was positively associated with SCD1/FADS2 levels and the oncogenic capacities of OvCa cells. In contrast, pharmaceutical inhibition and genetic ablation of SCD1/FADS2 retarded tumor growth, cancer stem cell (CSC) formation and reduced platinum resistance. Inhibition of SCD1/FADS2 directly downregulated GPX4 and the GSH/GSSG ratio, causing disruption of the cellular/mitochondrial redox balance and subsequently, iron-mediated lipid peroxidation and mitochondrial dysfunction in ascites-derived OvCa cells. Conclusions: Combinational treatment with SCD1/FADS2 inhibitors and cisplatin synergistically repressed tumor cell dissemination, providing a promising chemotherapeutic strategy against EOC peritoneal metastases.

Keywords:  lipid desaturases; lipid metabolism; ovarian cancer; oxidative stress; peritoneal metastases

DOI:  https://doi.org/10.7150/thno.70194
Lipids Health Dis. 2022 May 10. 21(1): 42

A multimodal analytical method to simultaneously determine monoacetyldiacylglycerols, medium and long chain triglycerides in biological samples during routine lipidomics.

Charles F Manful, Thu H Pham, Heather Spicer, Raymond H Thomas.

   BACKGROUND: Monoacetyldiglycerides (MAcDG), are acetylated triglycerides (TG) and an emerging class of bioactive or functional lipid with promising nutritional, medical, and industrial applications. A major challenge exists when analyzing MAcDG from other subclasses of TG in biological matrices, limiting knowledge on their applications and metabolism.
METHODS: Herein a multimodal analytical method for resolution, identification, and quantitation of MAcDG in biological samples was demonstrated based on thin layer chromatography-flame ionization detection complimentary with C30-reversed phase liquid chromatography-high resolution accurate mass tandem mass spectrometry. This method was then applied to determine the MAcDG molecular species composition and quantity in E. solidaginis larvae. The statistical method for analysis of TG subclass composition and molecular species composition of E. solidaginis larvae was one-way analysis of variance (ANOVA).
RESULTS: The findings suggest that the proposed analytical method could simultaneously provide a fast, accurate, sensitive, high throughput analysis of MAcDG from other TG subclasses, including the fatty acids, isomers, and molecular species composition.
CONCLUSION: This method would allow for MAcDG to be included during routine lipidomics analysis of biological samples and will have broad interests and applications in the scientific community in areas such as nutrition, climate change, medicine and biofuel innovations.

Keywords:  Acylated triglyceride; Eurosta solidaginis; Functional lipids; Liquid chromatography; Mass spectrometry; Thin layer chromatography; Triglyceride analysis

DOI:  https://doi.org/10.1186/s12944-022-01650-w
Small Methods. 2022 May 08. e2200130

Precise Metabolomics Reveals a Diversity of Aging-Associated Metabolic Features.

He Tian, Zhen Ni, Sin Man Lam, Wenxi Jiang, Fengjuan Li, Jie Du, Yuan Wang, Guanghou Shui.

  Mass spectrometry-based metabolomics has emerged as a powerful technique for biomedical research, although technical issues with its analytical precision and structural characterization remain. Herein, a robust non-targeted strategy for accurate quantitation and precise profiling of metabolomes is developed and applied to investigate plasma metabolic features associated with human aging. A comprehensive set of isotope-labeled standards (ISs) covering major metabolic pathways is incorporated to quantify polar metabolites. Matching rules to select ISs for calibration follow a primary criterion of minimal coefficients of variations (COVs). If minimal COVs between specific ISs for a particular metabolite fall within 5% window, a further selection of ISs is conducted based on structural similarities and proximity in retention time. The introduction and refined selection of appropriate ISs for quantitation reduces the COVs of 480 identified metabolites in quality control samples from 14.3% to 9.8% and facilitates identification of additional metabolite. Finally, the precise metabolomics approach reveals perturbations in a diverse array of metabolic pathways across aging that principally implicate steroid metabolism, amino acid metabolism, lipid metabolism, and purine metabolism, which allows the authors to draw correlates to the pathology of various age-related diseases. These findings provide clues for the prevention and treatment of these age-related diseases.

Keywords:  aging; precise quantification; untargeted metabolomics

DOI:  https://doi.org/10.1002/smtd.202200130
Trends Biotechnol. 2022 May 10. pii: S0167-7799(22)00102-0. [Epub ahead of print]

Single-cell mass spectrometry.

Mohammad Tajik, Mahroo Baharfar, William A Donald.

  Owing to recent advances in mass spectrometry (MS), tens to hundreds of proteins, lipids, and small molecules can be measured in single cells. The ability to characterize the molecular heterogeneity of individual cells is necessary to define the full assortment of cell subtypes and identify their function. We review single-cell MS including high-throughput, targeted, mass cytometry-based approaches and antibody-free methods for broad profiling of the proteome and metabolome of single cells. The advantages and disadvantages of different methods are discussed, as well as the challenges and opportunities for further improvements in single-cell MS. These methods is being used in biomedicine in several applications including revealing tumor heterogeneity and high-content drug screening.

Keywords:  cellular heterogeneity; lipidomics; mass cytometry; mass spectrometry; metabolomics; proteomics; single-cell analysis

DOI:  https://doi.org/10.1016/j.tibtech.2022.04.004
Bioinformatics. 2022 May 13. 38(10): 2872-2879

Improving confidence in lipidomic annotations by incorporating empirical ion mobility regression analysis and chemical class prediction.

Bailey S Rose, Jody C May, Jaqueline A Picache, Simona G Codreanu, Stacy D Sherrod, John A McLean.

MOTIVATION: Mass spectrometry-based untargeted lipidomics aims to globally characterize the lipids and lipid-like molecules in biological systems. Ion mobility increases coverage and confidence by offering an additional dimension of separation and a highly reproducible metric for feature annotation, the collision cross-section (CCS).
RESULTS: We present a data processing workflow to increase confidence in molecular class annotations based on CCS values. This approach uses class-specific regression models built from a standardized CCS repository (the Unified CCS Compendium) in a parallel scheme that combines a new annotation filtering approach with a machine learning class prediction strategy. In a proof-of-concept study using murine brain lipid extracts, 883 lipids were assigned higher confidence identifications using the filtering approach, which reduced the tentative candidate lists by over 50% on average. An additional 192 unannotated compounds were assigned a predicted chemical class.
AVAILABILITY AND IMPLEMENTATION: All relevant source code is available at https://github.com/McLeanResearchGroup/CCS-filter.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac197
J Inherit Metab Dis. 2022 May 11.

Application of Metabolite Set Enrichment Analysis (MSEA) on untargeted metabolomics data prioritizes relevant pathways and detects novel biomarkers for inherited metabolic disorders.

Brechtje Hoegen, Juliet E Hampstead, Udo F Engelke, Purva Kulkarni, Ron A Wevers, Han G Brunner, Karlien L M Coene, Christian Gilissen.

  Untargeted metabolomics (UM) allows for the simultaneous measurement of hundreds of metabolites in a single analytical run. The sheer amount of data generated in UM hampers its use in patient diagnostics because manual interpretation of all features is not feasible. Here, we describe the application of a pathway-based metabolite set enrichment analysis method (MSEA) to prioritise relevant biological pathways in UM data. We validate our method on a set of 55 patient samples with a diagnosed inherited metabolic disorder (IMD), and show that it complements feature-based prioritisation of biomarkers by placing the features in a biological context. In addition, we find that by taking enriched pathways shared across different IMDs we can identify common drugs and compounds that could otherwise obscure genuine disease biomarkers in an enrichment method. Finally, we demonstrate the potential of this method to identify novel candidate biomarkers for known IMDs. Our results show the added value of pathway-based interpretation of UM data in IMD diagnostics context.

Keywords:  Biochemical pathways; Biomarkers; Cystathionine ß-synthase; Inborn errors of metabolism; Inherited metabolic disorders; Mass spectrometry; Metabolite set enrichment analysis; Next generation metabolic screening; Untargeted metabolomics

DOI:  https://doi.org/10.1002/jimd.12522
Anal Chim Acta. 2022 May 22. pii: S0003-2670(22)00385-3. [Epub ahead of print]1208 339814

Mass defect filter technique combined with stable isotope tracing for drug metabolite identification using high-resolution mass spectrometry.

Chien-Yu Su, Jie-Huei Wang, Tien-Yi Chang, Chia-Lung Shih.

  Metabolism studies are one of the important steps in pharmaceutical research. LC-MS combined with metabolomics data-processing approaches have been developed for rapid screening of drug metabolites. Mass defect filter (MDF) is one of the LC/MS-based metabolomics data processing approaches and has been applied to screen drug metabolites. Although MDF can remove most interference ions from an incubation sample, the true positive rate of the retaining ions is relatively low (approximately 10%). To improve the efficacy of MDF, we developed a two-stage data-processing approach by combining MDF and stable isotope tracing (SIT) for metabolite identification. Pioglitazone (PIO), which is an antidiabetic drug used to treat type 2 diabetes mellitus, was taken as an example drug. Our results demonstrated that this new approach could substantially increase the validated rate from about 10% to 74%. Most of these validated metabolite signals (13/14) could be verified as PIO structure-related metabolites. In addition, we applied this approach to identify uncommon metabolite signals (a mass change beyond the window of 50 Da around its parent drug, MDF1). SIT could remove most interference ions (approximately 98%) identified by MDF1, and four out of five validated metabolite signals could be verified as PIO structure-related metabolites. Interestingly, a lot of the verified metabolites (10/17) were novel PIO metabolites. Among these novel metabolites, nine were thiazolidinedione ring-opening signals that might be related to the toxicity of PIO. Our developed approach could significantly improve the efficacy in drug metabolite identification compared with that of MDF.

Keywords:  High-resolution mass spectrometry; Mass defect filter; Pioglitazone; Stable isotope tracing

DOI:  https://doi.org/10.1016/j.aca.2022.339814
FASEB J. 2022 May;36 Suppl 1

Metabolic outliers in human disease.

Ralph DeBerardinis.

Many human diseases are caused by mutations that perturb metabolism at the cellular level and result in tissue dysfunction. Some metabolic perturbations result in disease by interrupting the canonical functions of the metabolic network: producing energy, generating precursors for macromolecular synthesis, maintaining redox balance, disposing of waste, etc. Others interfere with processes beyond the conventional metabolic network, interfering with signaling and gene expression networks. Understanding these pathological states of metabolic perturbation may help us develop rational approaches to normalize metabolism and restore health. We study two types of diseases characterized by metabolic dysfunction: inborn errors of metabolism and cancer. I will discuss ongoing work in these diseases that seeks to characterize abnormal metabolic states directly in human subjects, then uses experimental models to explore disease mechanisms and propose potential therapies. I will emphasize methods in metabolomics and stable isotope tracing that allow us observe metabolic phenotypes in intact systems relevant to physiology and disease, highlighting recent work on tumor metabolism in patients and genetically-defined metabolic anomalies that interfere with mammalian developmental programs.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I180
FASEB J. 2022 May;36 Suppl 1

Lipid Metabolism and Ferroptosis.

Scott Dixon.

Ferroptosis is an oxidative, iron-dependent form of non-apoptotic cell death that contributes to several forms of pathology and may be exploitable for cancer therapy. Ferroptosis can be induced using small molecule inhibitors of the plasma membrane cystine/glutamate antiporter system xc - or the glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4). The execution of ferroptosis requires membrane lipid peroxidation, but specific lipids and lipid metabolic enzymes that are involved in this process are only partly characterized. How ferroptosis sensitivity relates to other fundamental cellular processes such as the cell cycle is also unclear. We find that inhibition of the Rb-E2F cell cycle pathway causes downregulation of specific lipid metabolic enzymes, leading to an increase in polyunsaturated phospholipid abundance. In turn, this metabolic alteration specifically enhances ferroptosis sensitivity. Drugs that inhibit Rb-E2F pathway function synergize with a ferroptosis-inducing small molecule to arrest tumor growth in vivo. These results suggest that ferroptosis sensitivity may be linked to cell cycle progression through specific alterations in membrane phospholipid composition.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.0I209
Anal Chem. 2022 May 12.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Yixuan Xie, Kevin A Janssen, Alessandro Scacchetti, Elizabeth G Porter, Zongtao Lin, Roberto Bonasio, Benjamin A Garcia.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

DOI: https://doi.org/10.1021/acs.analchem.2c00471
FASEB J. 2022 May;36 Suppl 1

Lipin Expression Correlates to Differences in Phospholipid Profiles in Drug-Resistant Prostate Cancer Cells.

Morgan C Finnerty, Brian S Cummings.

PURPOSE: The association between clinical outcomes of prostate cancer progression and lipid remodeling is not well understood. Although it is known that increases in select circulating lipids correlate to decreased patient survival, the mechanisms mediating alterations in these lipids are not fully explained. We addressed this gap-in-knowledge using in vitro models of non-cancerous, hormone-sensitive, castration-resistant prostate cancer (CRPC), and docetaxel resistant (DR) CRPC cell lines combined with quantitative HPLC-ESI-Orbitrap-MS lipidomic analysis.
HYPOTHESIS: We hypothesized that the expression of lipin (phosphatidic acid phosphatase), an enzyme mediating the conversion of phosphatidic acid to diacylglycerol, correlates to changes in lipidomic profiles in prostate cancer cells. We further hypothesized that the lipidomic profile of metastatic CRPC cell lines (PC-3 and DU-145) will shift as they are undergoing multiple treatments of docetaxel.
METHODS: Lipids were isolated using the Bligh-Dyer extraction from non-cancerous, hormone-sensitive, CRPC, and DR-CRPC cell lines. Lipids were initially analyzed using an untargeted shotgun approach (ESI-MS), followed by a targeted-based quantitative HPLC-ESI-Orbitrap-MS approach. Post data acquisition were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency-based corrections. Online resources and software's utilized included MetaboAnalyst, LIPIDMATCH, LIPID MAPS, XCMS, and MZmine. Lipin-1 expression in cells was determined using immunoblot analysis. Docetaxel-sensitive traits were obtained after five treatments of 1 nM docetaxel between passages of the CRPC cell line PC-3. Results were demonstrated by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.
RESULTS: Our data showed significant changes in the abundance of lipid species between the various cell lines and a correlation between select lipid species and the progression of prostate cancer. Prominent among these were the levels of phosphatidic acid (PA) and diacylglycerol (DAG). Most notably, PA levels in cells tended to inversely correlate with DAG levels. These levels correlated to the decreased expression on lipin, specifically lipin-1.
CONCLUSIONS: Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, as compared to non-cancer cells. The protein expression of lipin-1 in most prostate cell lines correlated to the abundance of PA, excluding the DR cell lines. This suggests that altered lipin expression correlates to decreased survival. Additionally, dosing the parent and treated PC-3 cells with a range of docetaxel indicated that the 5x1nM treated cells had a larger cytotoxic effect to docetaxel, as compared to the non-treated cells.
REFERENCES: Ingram, L.M., et al., Identification of lipidomic profiles associated with drug-resistant prostate cancer cells. Lipids Health Dis, 2021. 20(1): p. 15.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3533
FASEB J. 2022 May;36 Suppl 1

Mass Spectrometry Based Subcellular Coenzyme Analysis.

Nathaniel W Snyder, Anna Bostwick, Hannah Pepper, Sophie Trefely.

Metabolism in eukaryotes relies on compartmentalization of processes between sub-cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub-cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture - Subcellular Fractionation (SILEC-SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13 C,15 N-label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography-high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R6113
Cells. 2022 Apr 23. pii: 1433. [Epub ahead of print]11(9):

The Role of Extracellular Vesicles in Metabolic Reprogramming of the Tumor Microenvironment.

Eran S Fridman, Lana Ginini, Ziv Gil.

  The tumor microenvironment (TME) includes a network of cancerous and non-cancerous cells, together with associated blood vessels, the extracellular matrix, and signaling molecules. The TME contributes to cancer progression during various phases of tumorigenesis, and interactions that take place within the TME have become targets of focus in cancer therapy development. Extracellular vesicles (EVs) are known to be conveyors of genetic material, proteins, and lipids within the TME. One of the hallmarks of cancer is its ability to reprogram metabolism to sustain cell growth and proliferation in a stringent environment. In this review, we provide an overview of TME EV involvement in the metabolic reprogramming of cancer and stromal cells, which favors cancer progression by enhancing angiogenesis, proliferation, metastasis, treatment resistance, and immunoevasion. Targeting the communication mechanisms and systems utilized by TME-EVs is opening a new frontier in cancer therapy.

Keywords:  cancer metabolism; exosomes; extracellular vesicles (EVs); glycolysis; tumor microenvironment (TME)

DOI:  https://doi.org/10.3390/cells11091433
Nutrients. 2022 Apr 21. pii: 1722. [Epub ahead of print]14(9):

Exploring the Role of Metabolites in Cancer and the Associated Nerve Crosstalk.

Inah Gu, Emory Gregory, Casey Atwood, Sun-Ok Lee, Young Hye Song.

  Since Otto Warburg's first report on the increased uptake of glucose and lactate release by cancer cells, dysregulated metabolism has been acknowledged as a hallmark of cancer that promotes proliferation and metastasis. Over the last century, studies have shown that cancer metabolism is complex, and by-products of glucose and glutamine catabolism induce a cascade of both pro- and antitumorigenic processes. Some vitamins, which have traditionally been praised for preventing and inhibiting the proliferation of cancer cells, have also been proven to cause cancer progression in a dose-dependent manner. Importantly, recent findings have shown that the nervous system is a key player in tumor growth and metastasis via perineural invasion and tumor innervation. However, the link between cancer-nerve crosstalk and tumor metabolism remains unclear. Here, we discuss the roles of relatively underappreciated metabolites in cancer-nerve crosstalk, including lactate, vitamins, and amino acids, and propose the investigation of nutrients in cancer-nerve crosstalk based on their tumorigenicity and neuroregulatory capabilities. Continued research into the metabolic regulation of cancer-nerve crosstalk will provide a more comprehensive understanding of tumor mechanisms and may lead to the identification of potential targets for future cancer therapies.

Keywords:  amino acid metabolism; cancer; cancer–nerve crosstalk; lactate; metabolites; perineural invasion; tumor innervation; vitamins

DOI:  https://doi.org/10.3390/nu14091722
Anal Chem. 2022 May 11.

An Organ-Specific Metabolite Annotation Approach for Ambient Mass Spectrometry Imaging Reveals Spatial Metabolic Alterations of a Whole Mouse Body.

Ying Zhu, Qingce Zang, Zhigang Luo, Jiuming He, Ruiping Zhang, Zeper Abliz.

Rapid and accurate metabolite annotation in mass spectrometry imaging (MSI) can improve the efficiency of spatially resolved metabolomics studies and accelerate the discovery of reliable in situ disease biomarkers. To date, metabolite annotation tools in MSI generally utilize isotopic patterns, but high-throughput fragmentation-based identification and biological and technical factors that influence structure elucidation are active challenges. Here, we proposed an organ-specific, metabolite-database-driven approach to facilitate efficient and accurate MSI metabolite annotation. Using data-dependent acquisition (DDA) in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to generate high-coverage product ions, we identified 1620 unique metabolites from eight mouse organs (brain, liver, kidney, heart, spleen, lung, muscle, and pancreas) and serum. Following the evaluation of the adduct form difference of metabolite ions between LC-MS and airflow-assisted desorption electrospray ionization (AFADESI)-MSI and deciphering organ-specific metabolites, we constructed a metabolite database for MSI consisting of 27,407 adduct ions. An automated annotation tool, MSIannotator, was then created to conduct metabolite annotation in the MSI dataset with high efficiency and confidence. We applied this approach to profile the spatially resolved landscape of the whole mouse body and discovered that metabolites were distributed across the body in an organ-specific manner, which even spanned different mouse strains. Furthermore, the spatial metabolic alteration in diabetic mice was delineated across different organs, exhibiting that differentially expressed metabolites were mainly located in the liver, brain, and kidney, and the alanine, aspartate, and glutamate metabolism pathway was simultaneously altered in these three organs. This approach not only enables robust metabolite annotation and visualization on a body-wide level but also provides a valuable database resource for underlying organ-specific metabolic mechanisms.

DOI: https://doi.org/10.1021/acs.analchem.2c00557
FASEB J. 2022 May;36 Suppl 1

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration.

Chaylen J Andolino, Dorothy Teegarden.

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non-metastatic MCF10A-ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration-a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A-ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14 C-palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13 C-acetate (60%), 13 C-glucose (65%), and 13 C-glutamine (50%) into palmitate compared to the MCF10A-ras cells (LC-MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly-labeled 13 C-glucose and 13 C-glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A-ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A-ras cells, as determined by inhibiting the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound-healing assays). Pretreatment with the FASN inhibitor TVB-3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle-treated cells. Similarly, pretreatment with inhibitors of the TAG-synthesizing enzymes diacylglycerol O-acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO-dependent migration.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R2040
Cancer Res. 2022 May 10. pii: canres.4256.2021. [Epub ahead of print]

Hypoxia-reoxygenation couples 3βHSD1 enzyme and cofactor upregulation to facilitate androgen biosynthesis and hormone therapy resistance in prostate cancer.

Liang Qin, Yoon-Mi Chung, Michael Berk, Bryan Naelitz, Ziqi Zhu, Eric Klein, Abhishek A Chakraborty, Nima Sharifi.

Androgen deprivation therapy suppresses tumor androgen receptor (AR) signaling by depleting circulating testosterone and is a mainstay treatment for advanced prostate cancer. Despite initial treatment response, castration-resistant prostate cancer nearly always develops and remains driven primarily by the androgen axis. Here we investigated how changes in oxygenation affect androgen synthesis. In prostate cancer cells, chronic hypoxia coupled to reoxygenation resulted in efficient metabolism of androgen precursors to produce androgens and activate AR. Hypoxia induced 3βHSD1, the rate-limiting androgen synthesis regulator, and reoxygenation replenished necessary cofactors, suggesting that hypoxia and reoxygenation both facilitate potent androgen synthesis. The EGLN1/VHL/HIF2α pathway induced 3βHSD1 expression through direct binding of HIF2α to the 5' regulatory region of HSD3B1 to promote transcription. Overexpression of HIF2α facilitated prostate cancer progression, which largely depended on 3βHSD1. Inhibition of HIF2α with the small molecule PT2399 prevented prostate cancer cell proliferation. These results thus identify HIF2α as a regulator of androgen synthesis and potential therapeutic target in prostate cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-4256