1 H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae.

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using 1 H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.

Enhanced OXPHOS, glutaminolysis and ß-oxidation constitute the metastatic phenotype of melanoma cells.

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Epigenetic modifications, chromatin distribution and TP53 transcription in a model of breast cancer progression.

In the present paper we aimed to characterize epigenetic aspects and analyze TP53 transcription in the 21 T series, composed of breast cell lines: non-cancerous H16N2; Atypical Ductal Hyperplasia 21PT; Ductal Carcinoma in situ 21NT and Invasive Metastatic Carcinoma 21MT1. We detected a global genomic hypomethylation in 21NT and 21MT1. The histone modification markers analysis showed an important global decrease of the active chromatin mark H4Ac in 21MT1 relative to the other cell lines while the repressive mark H3K9Me3 were not significantly altered. The mRNA levels of DNA methylation and histone modification key enzymes are consistent with the observed genomic hypomethylation and histone hypoacetylation. The expression of DNMT3A/B increased at the initial stages of oncogenesis and the expression of DNMT1 and HAT1 decreased at the advanced stages of breast cancer. Using a confocal immunofluorescent assay, we observed that H4Ac was mostly located at the periphery and the repressive mark H3K9Me3, at the center of 21NT and 21MT1 cells nuclei. TP53 P1 promoter was found to be in an open chromatin state, with a relatively high enrichment of H4Ac and similar TP53 transcription levels in all 21 T cell lines. In conclusion, we observed epigenetic alterations (global genome hypomethylation, global hypoacetylation and accumulation of pericentric heterochromatin) in metastatic breast cancer cells of the 21 T series. These alterations may act at later stages of breast cancer progression and may not affect TP53 transcription at the P1 promoter.

Integrated NMR and MS Analysis of the Plasma Metabolome Reveals Major Changes in One-Carbon, Lipid, and Amino Acid Metabolism in Severe and Fatal Cases of COVID-19.

Brazil has the second-highest COVID-19 death rate worldwide, and Rio de Janeiro is among the states with the highest rate in the country. Although vaccine coverage has been achieved, it is anticipated that COVID-19 will transition into an endemic disease. It is concerning that the molecular mechanisms underlying clinical evolution from mild to severe disease, as well as the mechanisms leading to long COVID-19, are not yet fully understood. NMR and MS-based metabolomics were used to identify metabolites associated with COVID-19 pathophysiology and disease outcome. Severe COVID-19 cases (n = 35) were enrolled in two reference centers in Rio de Janeiro within 72 h of ICU admission, alongside 12 non-infected control subjects. COVID-19 patients were grouped into survivors (n = 18) and non-survivors (n = 17). Choline-related metabolites, serine, glycine, and betaine, were reduced in severe COVID-19, indicating dysregulation in methyl donors. Non-survivors had higher levels of creatine/creatinine, 4-hydroxyproline, gluconic acid, and N-acetylserine, indicating liver and kidney dysfunction. Several changes were greater in women; thus, patients' sex should be considered in pandemic surveillance to achieve better disease stratification and improve outcomes. These metabolic alterations may be useful to monitor organ (dys) function and to understand the pathophysiology of acute and possibly post-acute COVID-19 syndromes.

Loop domain organization of the p53 locus in normal and breast cancer cells correlates with the transcriptional status of the TP53 and the neighboring genes.

P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines. We have found that in the control breast epithelial cell line, HB2, the TP53 gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231, and BT474, in which the domain size was increased and TP53 transcription was decreased. We propose a model in which the organization of the TP53 gene domain correlates with the transcriptional status of TP53 and neighboring genes.

Metabolic Profile of Oral Squamous Carcinoma Cell Lines Relies on a Higher Demand of Lipid Metabolism in Metastatic Cells.

Tumor cells are subjected to a broad range of selective pressures. As a result of the imposed stress, subpopulations of surviving cells exhibit individual biochemical phenotypes that reflect metabolic reprograming. The present work aimed at investigating metabolic parameters of cells displaying increasing degrees of metastatic potential. The metabolites present in cell extracts fraction of tongue fibroblasts and of cell lines derived from human tongue squamous cell carcinoma lineages displaying increasing metastatic potential (SCC9 ZsG, LN1 and LN2) were analyzed by 1H NMR (nuclear magnetic resonance) spectroscopy. Living, intact cells were also examined by the non-invasive method of fluorescence lifetime imaging microscopy (FLIM) based on the auto fluorescence of endogenous NADH. The cell lines reproducibly exhibited distinct metabolic profiles confirmed by Partial Least-Square Discriminant Analysis (PLS-DA) of the spectra. Measurement of endogenous free and bound NAD(P)H relative concentrations in the intact cell lines showed that ZsG and LN1 cells displayed high heterogeneity in the energy metabolism, indicating that the cells would oscillate between glycolysis and oxidative metabolism depending on the microenvironment's composition. However, LN2 cells appeared to have more contributions to the oxidative status, displaying a lower NAD(P)H free/bound ratio. Functional experiments of energy metabolism, mitochondrial physiology, and proliferation assays revealed that all lineages exhibited similar energy features, although resorting to different bioenergetics strategies to face metabolic demands. These differentiated functions may also promote metastasis. We propose that lipid metabolism is related to the increased invasiveness as a result of the accumulation of malonate, methyl malonic acid, n-acetyl and unsaturated fatty acids (CH2)n in parallel with the metastatic potential progression, thus suggesting that the NAD(P)H reflected the lipid catabolic/anabolic pathways.