bims-mascan 2022-10-09 papers

Issue of 2022‒10‒09
eight papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

Front Oncol. 2022 ;12 1011191

Differential glutamine metabolism in the tumor microenvironment - studies in diversity and heterogeneity: A mini-review.

Michael D Claiborne, Robert Leone.

Increased glutamine metabolism is a hallmark of many cancer types. In recent years, our understanding of the distinct and diverse metabolic pathways through which glutamine can be utilized has grown more refined. Additionally, the different metabolic requirements of the diverse array of cell types within the tumor microenvironment complicate the strategy of targeting any particular glutamine pathway as cancer therapy. In this Mini-Review, we discuss recent advances in further clarifying the cellular fate of glutamine through different metabolic pathways. We further discuss potential promising strategies which exploit the different requirements of cells in the tumor microenvironment as it pertains to glutamine metabolism in an attempt to suppress cancer growth and enhance anti-tumor immune responses.

Keywords: cancer; glutamine; immunooncology; metabolism; tumor microenvironment

DOI: https://doi.org/10.3389/fonc.2022.1011191

Cancer Metab. 2022 Oct 03. 10(1): 14

ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma.

Timothy D Klasson, Edward L LaGory, Hongjuan Zhao, Star K Huynh, Ioanna Papandreou, Eui Jung Moon, Amato J Giaccia.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood.METHODS: We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed.
RESULTS: We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC.
CONCLUSIONS: ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

Keywords: 5-lipoxygenase (5-LOX); Acyl-CoA synthetase 3 (ACSL3); Clear cell renal cell carcinoma (ccRCC); Ferroptosis; Lipid droplets; Lipid metabolism

DOI: https://doi.org/10.1186/s40170-022-00290-z

Anticancer Res. 2022 Oct;42(10): 5077-5081

Synthesis of 13C Labeled Vitamin D Metabolites for Their Use in LC-MS/MS: Valuable Tools for Cancer Research and Other Applications.

Lars Kattner, Erik Rauch.

BACKGROUND/AIM: Simultaneous assessment of various vitamin D metabolites in human biofluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) represents a new promising tool for the differential diagnosis of vitamin D-related diseases. Particularly, besides 25(OH)VD2/3, low-abundant medicinally relevant vitamin D metabolites, such as 24,25(OH)2VD2/3, 1,25(OH)2VD2/3, and 1,24,25(OH)3VD2/3, along with their 3-epi-derivatives have to be considered.MATERIALS AND METHODS: The assessment of these metabolites by LC-MS/MS requires the development of calibration and reference standards, that is, their labeling with multiple deuterium-, or even better, 13C- atoms.
RESULTS: Overall, 10 13C-labeled vitamin D metabolites have been chemically synthesized and obtained in good yield and high purity.
CONCLUSION: Access to a wide variety of 13C-labeled highly pure vitamin D metabolites enables the advancement of LC-MS/MS applications towards a better understanding of differential diagnosis of vitamin D-related diseases.

Keywords: Cancer prevention; LC-MS/MS assay development; vitamin D metabolites; vitamin D synthesis

DOI: https://doi.org/10.21873/anticanres.16016

Anal Chem. 2022 Oct 04.

On-capillary Cell Lysis Enables Top-down Proteomic Analysis of Single Mammalian Cells by CE-MS/MS.

Kendall R Johnson, Yunfan Gao, Michal Greguš, Alexander R Ivanov.

Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (∼1000 cells) and demonstrated 0-9 intact cells injected with a dependency on the duration of spray voltage application. Spray voltage applied for 2 min injected an average of 7 ± 2 cells and resulted in 33-57 protein and 40-88 proteoform identifications (N = 4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single HeLa cells (N = 4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17-40 proteins and 23-50 proteoforms identified. An additional cell line, THP-1, was analyzed at the single-cell level, and proteoform abundances were compared to show the capabilities of single-cell TDP (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.

DOI: https://doi.org/10.1021/acs.analchem.2c03045

J Proteome Res. 2022 Oct 06.

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides.

Helga Hevér, Kinga Nagy, Andrea Xue, Simon Sugár, Kinga Komka, Károly Vékey, László Drahos, Ágnes Révész.

Identification and characterization of N-glycopeptides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument. The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10-50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

Keywords: N-glycosylation; bottom-up proteomics; collision energy optimization; glycopeptide fragmentation; identification score; search engine; tandem mass spectrometry; transferability

DOI: https://doi.org/10.1021/acs.jproteome.2c00519

Proteomics. 2022 Oct 03. e2200271

iBench: A ground truth approach for advanced validation of mass spectrometry identification method.

John A Cormican, Wai Tuck Soh, Michele Mishto, Juliane Liepe.

The discovery of many noncanonical peptides detectable with sensitive mass spectrometry inside, outside and on cells shepherded the development of novel methods for their identification, often not supported by a systematic benchmarking with other methods. We here propose iBench, a bioinformatic tool that can construct ground truth proteomics datasets and cognate databases, thereby generating a training court wherein methods, search engines, and proteomics strategies can be tested, and their performances estimated by the same tool. iBench can be coupled to the main database search engines, allows the selection of customized features of mass spectrometry spectra and peptides, provides standard benchmarking outputs, and is open source. The proof-of-concept application to tryptic proteome digestions, immunopeptidomes and synthetic peptide libraries dissected the impact that noncanonical peptides could have on the identification of canonical peptides by Mascot search with rescoring via Percolator (Mascot+Percolator). This article is protected by copyright. All rights reserved.

Keywords: HLA; benchmarking; immunopeptidome; method performance; proteomics

DOI: https://doi.org/10.1002/pmic.202200271

Cell Rep. 2022 Oct 04. pii: S2211-1247(22)01286-4. [Epub ahead of print]41(1): 111445

MCL-1 is a master regulator of cancer dependency on fatty acid oxidation.

Michelle S Prew, Utsarga Adhikary, Dong Wook Choi, Erika P Portero, Joao A Paulo, Pruthvi Gowda, Amit Budhraja, Joseph T Opferman, Steven P Gygi, Nika N Danial, Loren D Walensky.

MCL-1 is an anti-apoptotic BCL-2 family protein essential for survival of diverse cell types and is a major driver of cancer and chemoresistance. The mechanistic basis for the oncogenic supremacy of MCL-1 among its anti-apoptotic homologs is unclear and implicates physiologic roles of MCL-1 beyond apoptotic suppression. Here we find that MCL-1-dependent hematologic cancer cells specifically rely on fatty acid oxidation (FAO) as a fuel source because of metabolic wiring enforced by MCL-1 itself. We demonstrate that FAO regulation by MCL-1 is independent of its anti-apoptotic activity, based on metabolomic, proteomic, and genomic profiling of MCL-1-dependent leukemia cells lacking an intact apoptotic pathway. Genetic deletion of Mcl-1 results in transcriptional downregulation of FAO pathway proteins such that glucose withdrawal triggers cell death despite apoptotic blockade. Our data reveal that MCL-1 is a master regulator of FAO, rendering MCL-1-driven cancer cells uniquely susceptible to treatment with FAO inhibitors.

Keywords: BCL-2 family; CP: Cancer; CP: Metabolism; MCL-1; apoptosis; cancer; fatty acid oxidation; metabolism

DOI: https://doi.org/10.1016/j.celrep.2022.111445