The analysis of biological samples in untargeted metabolomic studies using LC-MS yields tens of thousands of ion signals. Annotating these features is of the utmost importance for answering questions as fundamental as, e.g. how many metabolites are there in a given sample. Here, we introduce CliqueMS, a new algorithm for annotating in-source LC-MS1 data. CliqueMS is based on the similarity between coelution profiles and therefore, as opposed to most methods, allows for the annotation of a single spectrum. Furthermore, CliqueMS improves upon the state of the art in several dimensions: (i) it uses a more discriminatory feature similarity metric; (ii) it treats the similarities between features in a transparent way by means of a simple generative model; (iii) it uses a well-grounded maximum likelihood inference approach to group features; (iv) it uses empirical adduct frequencies to identify the parental mass and (v) it deals more flexibly with the identification of the parental mass by proposing and ranking alternative annotations. We validate our approach with simple mixtures of standards and with real complex biological samples. CliqueMS reduces the thousands of features typically obtained in complex samples to hundreds of metabolites, and it is able to correctly annotate more metabolites and adducts from a single spectrum than available tools. https://CRAN.R-project.org/package=cliqueMS and https://github.com/osenan/cliqueMS. Supplementary data are available at Bioinformatics online.

Transforming Growth Factor beta (TGF-β) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profile of hepatocellular carcinoma (HCC) cells that show differences in TGF-β expression. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without significant differences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fluxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-β function was activated with extracellular TGF-β1 or inhibited through TGF-β receptor I silencing showed that TGF-β induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-β also regulated the expression of key genes involved in the flux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-β pathway regulates oxidative metabolism in HCC cells.

Aims/hypothesis In mammals, the evolutionary conserved family of Mg²⁺-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. Methods The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. Results Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson–Golabi–Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. Conclusions/interpretation Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.

Anticancer therapies can induce cellular senescence or drug-tolerant persistence, two types of proliferative arrest that differ in their stability. While senescence is highly stable, persister cells efficiently resume proliferation upon therapy termination, resulting in tumor relapse. Here, we used an ATP-competitive mTOR inhibitor to induce and characterize persistence in human cancer cells of various origins. Using this model and previously described models of senescence, we compared the same cancer cell lines under the two types of proliferative arrest. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (senescence-associated secretory phenotype or SASP). A genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacologic inhibition of serine hydroxymethyltransferase, a key enzyme that feeds methyl groups from serine into 1C metabolism. Investigation into the relationship between 1C metabolism and the epigenetic regulation of transcription uncovered the presence of the repressive heterochromatic mark H4K20me3 at the promoters of SASP and IFN response genes in persister cells, whereas it was absent in senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study identifies distinctive features and actionable vulnerabilities of persister cancer cells and provides mechanistic insight into their low inflammatory activity. Significance: Cell persistence and senescence are distinct states of proliferative arrest induced by cancer therapy, with persister cells being characterized by the silencing of inflammatory genes through the heterochromatic mark H4K20me3. See related commentary by Schmitt, p. 7