Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines.

INTRODUCTION: KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. OBJECTIVES: To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. METHODS: Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/- and KRAS G13D/-. RESULTS: Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/- cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. CONCLUSIONS: Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS

Standardized nomenclature and open science in Human Genomics.

[Extract] Two recent papers have highlighted the vital importance of using standardized nomenclature in reporting data, especially when this is of clinical relevance. Higgins et al. [1] have drawn attention to the crucial matter of the appropriate nomenclature of DNA variants in scientific publications. It is critical that DNA variants can be identified unambiguously. And Fujiyoshi et al. [2] have called for gene products to be referenced using the approved gene symbol for the encoding gene, along with an appropriate database ID (HGNC ID, with UniProt ID where required, see Table 1 for resources for vertebrate genes). Confusion can impede data sharing and scientific progress, as well as potentially result in patient harm

Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines

Introduction: KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations. Objectives: To investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations. Methods: We applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants. Results: CRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism. Conclusions: Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS

Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of fmo5-/- mice, decreases plasma cholesterol and epididymal fat deposition

We previously showed that Fmo5−/− mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5−/− and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5−/− mice. Antibiotic-treatment of Fmo5−/− mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5−/− mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5−/− to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5−/− mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5−/− mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol

Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5-/- mice, decreases plasma cholesterol and epididymal fat deposition

We previously showed that Fmo5-/- mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5-/- and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5-/- mice. Antibiotic-treatment of Fmo5-/- mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5-/- mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5-/- to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5-/- mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5-/- mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol