The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast ...cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB-dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor-treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor-negative status, and shorter overall survival. Our results define an NF-κB-regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype.
Amino acid analysis is a powerful bioanalytical technique for many biomedical research endeavors, including cancer, emergency medicine, nutrition and neuroscience research. In the present study, we ...present a 3 min analytical method for underivatized amino acid analysis that employs ultra high-performance liquid chromatography and high-resolution quadrupole orbitrap mass spectrometry. This method has demonstrated linearity (mM to nM range), reproducibility (intra-day <5 %, inter-day <20 %), sensitivity (low fmol) and selectivity. Here, we illustrate the rapidity and accuracy of the method through comparison with conventional liquid chromatography–mass spectrometry methods. We further demonstrate the robustness and sensitivity of this method on a diverse range of biological matrices. Using this method we were able to selectively discriminate murine pancreatic cancer cells with and without knocked down expression of hypoxia-inducible factor 1α; plasma, lymph and bronchioalveolar lavage fluid samples from control versus hemorrhaged rats; and muscle tissue samples harvested from rats subjected to both low-fat and high-fat diets. Furthermore, we were able to exploit the sensitivity of the method to detect and quantify the release of glutamate from sparsely isolated murine taste buds. Spiked in light or heavy standards (¹³C₆-arginine, ¹³C₆-lysine, ¹³C ₅ ¹⁵ N₂-glutamine) or xenometabolites (5-fluorouracil) were used to determine coefficients of variation, confirm linearity of relative quantitation in four different matrices, and overcome matrix effects for absolute quantitation. The presented method enables high-throughput analysis of low-abundance samples requiring only one percent of the material extracted from 100,000 cells, 10 µl of biological fluid, or 2 mg of muscle tissue.
In this study we interrogated the metabolome of human acute myeloid leukemia (AML) stem cells to elucidate properties relevant to therapeutic intervention. We demonstrate that amino acid uptake, ...steady-state levels, and catabolism are all elevated in the leukemia stem cell (LSC) population. Furthermore, LSCs isolated from de novo AML patients are uniquely reliant on amino acid metabolism for oxidative phosphorylation and survival. Pharmacological inhibition of amino acid metabolism reduces oxidative phosphorylation and induces cell death. In contrast, LSCs obtained from relapsed AML patients are not reliant on amino acid metabolism due to their ability to compensate through increased fatty acid metabolism. These findings indicate that clinically relevant eradication of LSCs can be achieved with drugs that target LSC metabolic vulnerabilities.
•Leukemia stem cells (LSCs) rely on amino acids for survival•LSCs use amino acids to fuel oxidative phosphorylation (OXPHOS)•Venetoclax with azacitidine inhibits OXPHOS in LSCs from leukemia patients•Venetoclax with azacitidine targets OXPHOS by inhibiting amino acid metabolism
By interrogating metabolic properties of human acute myeloid leukemia, Jones et al. reveal increased amino acid metabolism in leukemia stem cells (LSCs), which rely on amino acids for oxidative phosphorylation and survival. Venetoclax with azacitidine induces LSC toxicity by decreasing amino acid uptake.
The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, ...red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
Metabolomics in transfusion medicine Nemkov, Travis; Hansen, Kirk C.; Dumont, Larry J. ...
Transfusion (Philadelphia, Pa.),
April 2016, Letnik:
56, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Biochemical investigations on the regulatory mechanisms of red blood cell (RBC) and platelet (PLT) metabolism have fostered a century of advances in the field of transfusion medicine. Owing to these ...advances, storage of RBCs and PLT concentrates has become a lifesaving practice in clinical and military settings. There, however, remains room for improvement, especially with regard to the introduction of novel storage and/or rejuvenation solutions, alternative cell processing strategies (e.g., pathogen inactivation technologies), and quality testing (e.g., evaluation of novel containers with alternative plasticizers). Recent advancements in mass spectrometry–based metabolomics and systems biology, the bioinformatics integration of omics data, promise to speed up the design and testing of innovative storage strategies developed to improve the quality, safety, and effectiveness of blood products.
Here we review the currently available metabolomics technologies and briefly describe the routine workflow for transfusion medicine–relevant studies. The goal is to provide transfusion medicine experts with adequate tools to navigate through the otherwise overwhelming amount of metabolomics data burgeoning in the field during the past few years.
Descriptive metabolomics data have represented the first step omics researchers have taken into the field of transfusion medicine. However, to up the ante, clinical and omics experts will need to merge their expertise to investigate correlative and mechanistic relationships among metabolic variables and transfusion‐relevant variables, such as 24‐hour in vivo recovery for transfused RBCs. Integration with systems biology models will potentially allow for in silico prediction of metabolic phenotypes, thus streamlining the design and testing of alternative storage strategies and/or solutions.
Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should ...identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.
Red blood cell transfusion is a life-saving intervention, and storage is a logistic necessity to make ~110 million units available for transfusion every year worldwide. However, storage in the blood ...bank is associated with a progressive metabolic decline, which correlates with the accumulation of morphological lesions, increased intra- and extra-vascular hemolysis upon transfusion, and altered oxygen binding/off-loading kinetics. Prior to storage, red blood cells are suspended in nutrient formulations known as additive solutions to prolong cellular viability. Despite a thorough expansion of knowledge regarding red blood cell biology over the past few decades, only a single new additive solution has been approved by the Food and Drug Administration this century, owing in part to the limited capacity for development of novel formulations. As a proof of principle, we leveraged a novel high-throughput metabolomics technology as a platform for rapid data-driven development and screening of novel additive solutions for blood storage under both normoxic and hypoxic conditions. To this end, we obtained leukocyte-filtered red blood cells (RBCs) and stored them under normoxic or hypoxic conditions in 96 well plates (containing polyvinylchloride plasticized with diethylhexylphthalate to concentrations comparable to full size storage units) in the presence of an additive solution supplemented with six different compounds. To inform this data-driven strategy, we relied on previously identified metabolic markers of the RBC storage lesion that associates with measures of hemolysis and post-transfusion recovery, which are the FDA gold standards to predict stored blood quality, as well as and metabolic predictors of oxygen binding/off-loading parameters. Direct quantitation of these predictors of RBC storage quality were used here-along with detailed pathway analysis of central energy and redox metabolism-as a decision-making tool to screen novel additive formulations in a multiplexed fashion. Candidate supplements are shown here that boost-specific pathways. These metabolic effects are only in part dependent on the SO
storage conditions. Through this platform, we anticipate testing thousands of novel additives and combinations thereof in the upcoming months.
Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small ...molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.
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