NMR Analysis of Carboxylate Isotopomers of 13C-Metabolites by Chemoselective Derivatization with 15N-Cholamine.

A substantial fraction of common metabolites contains carboxyl functional groups. Their 13C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the 13C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. 15N1-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via 15N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how 13C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine 15N atom in the derivatives. We have applied this method for the determination of 13C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.

RON-augmented cholesterol biosynthesis in breast cancer metastatic progression and recurrence.

Recurrence remains a significant clinical barrier to improving breast cancer patient outcomes. The RON receptor is a predictor of metastatic progression and recurrence in breast cancers of all subtypes. RON directed therapies are in development, but preclinical data directly testing the impact of RON inhibition on metastatic progression/recurrence are lacking, and mechanisms to exert this function remain unclear. Herein, we modeled breast cancer recurrence using implantation of RON-overexpressing murine breast cancer cells. Recurrent growth was examined after tumor resection via in vivo imaging and ex vivo culture of circulating tumor cells from whole blood samples from tumor bearing mice. In vitro functional assessment of was performed using mammosphere formation assays. Transcriptomic pathway enrichment identified glycolysis and cholesterol biosynthesis pathways, transcription factor targets, and signaling pathways enriched in RON-overexpressing breast cancer cells. BMS777607, a RON inhibitor, abrogated CTC colony formation tumor cells and tumor recurrence. RON promoted mammosphere formation through upregulated cholesterol production that utilizes glycolysis-derived substrates. In mouse models with RON overexpression, statin-mediated inhibition of cholesterol biosynthesis impeded metastatic progression and recurrence but does not affect the primary tumor. RON upregulates glycolysis and cholesterol biosynthesis gene expression by two pathways: MAPK-dependent c-Myc expression and ß-catenin -dependent SREBP2 expression.

NMR-based metabolomic analysis identifies RON-DEK-ß-catenin dependent metabolic pathways and a gene signature that stratifies breast cancer patient survival.

BACKGROUND: Advances in detection techniques and treatment have increased the diagnosis of breast cancer at early stages; however, recurrence occurs in all breast cancer subtypes, and both recurrent and de novo metastasis are typically treatment resistant. A growing body of evidence supports the notion that metabolic plasticity drives cancer recurrence. RON and DEK are proteins that promote cancer metastasis and synergize mechanistically to activate ß-catenin, but the metabolic consequences are unknown. METHODS: To ascertain RON-DEK-ß-catenin dependent metabolic pathways, we utilized an NMR-based metabolomics approach to determine steady state levels of metabolites. We also interrogated altered metabolic pathway gene expression for prognostic capacity in breast cancer patient relapse-free and distant metastasis-free survival and discover a metabolic signature that is likely associated with recurrence. RESULTS: RON-DEK-ß-catenin loss showed a consistent metabolite regulation of succinate and phosphocreatine. Consistent metabolite alterations between RON and DEK loss (but not ß-catenin) were found in media glucose consumption, lactate secretion, acetate secretion, and intracellular glutamine and glutathione levels. Consistent metabolite alterations between RON and ß-catenin loss (and not DEK) were found only in intracellular lactate levels. Further pathway hits include ß-catenin include glycolysis, glycosylation, TCA cycle/anaplerosis, NAD+ production, and creatine dynamics. Genes in these pathways epistatic to RON-DEK-ß-catenin were used to define a gene signature that prognosticates breast cancer patient survival and response to chemotherapy. CONCLUSIONS: The RON-DEK-ß-catenin axis regulates the numerous metabolic pathways with significant associations to breast cancer patient outcomes.

A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.

Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N2 in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110−115 °C to give >90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.

Elevated Circulatory Proline to Glutamine Ratio (PQR) in Endometriosis and Its Potential as a Diagnostic Biomarker.

Endometriosis (EM) is a hormone-dependent gynecological disease associated with chronic pelvic pain and altered immuno-inflammatory processes. It shares some cancer-like characteristics such as increased proline biosynthesis and activated glutaminolysis. Both proline and glutamine are interconvertible metabolically, and studies have shown their roles in cancer cell metabolic reprogramming, redox homeostasis, occurrence/development of endometrial carcinoma, and its further progression toward the malignant state. So based on this, we hypothesized that the circulatory proline to glutamine ratio (PQR) would be altered in EM and may serve as an indicative biomarker to improve the clinical diagnosis of EM. In present study, the circulatory-PQR levels were estimated for 39 EM patients and 48 age matched healthy female subjects using 800 MHz NMR spectroscopy. Among 39 EM patients, 15 were in the clinical stages I to II and referred to here as moderate EM (MEM) patients and 24 were in the clinical stages III to IV and referred here as severe EM (SEM) patients. The circulatory-PQR levels were significantly increased in EM patients (0.99 ± 0.13 µM in MEM; 1.39 ± 0.22 µM in SEM) compared to normal control (NC) subjects (0.52 ± 0.05 µM in NC). Further, the circulatory PQR levels exhibit the highest diagnostic potential with area under receiver operating characteristic (AUROC) curve values equal to 0.87 ± 0.04 [95%CI = 0.79-0.96] for MEM and 0.89 ± 0.04 [95% CI = 0.82-0.96] for SEM. These results suggested that circulatory-PQR has significant potential to serve as a noninvasive biomarker for diagnostic/prognostic screening of EM and further underscored the importance of these two nonessential amino acids (proline and glutamine) in cancer metabolism.

Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.

Vaginal metabolome: towards a minimally invasive diagnosis of microbial invasion of the amniotic cavity in women with preterm labor.

Microbial invasion of the amniotic cavity (MIAC) is only identified by amniocentesis, an invasive procedure that limits its clinical translation. Here, we aimed to evaluate whether the vaginal metabolome discriminates the presence/absence of MIAC in women with preterm labor (PTL) and intact membranes. We conducted a case-control study in women with symptoms of PTL below 34 weeks who underwent amniocentesis to discard MIAC. MIAC was defined as amniotic fluid positive for microorganisms identified by specific culture media. The cohort included 16 women with MIAC and 16 control (no MIAC). Both groups were matched for age and gestational age at admission. Vaginal fluid samples were collected shortly after amniocentesis. Metabolic profiles were analyzed by nuclear magnetic resonance (NMR) spectroscopy and compared using multivariate and univariate statistical analyses to identify significant differences between the two groups. The vaginal metabolomics profile of MIAC showed higher concentrations of hypoxanthine, proline, choline and acetylcholine and decreased concentrations of phenylalanine, glutamine, isoleucine, leucine and glycerophosphocholine. In conclusion, metabolic changes in the NMR-based vaginal metabolic profile are able to discriminate the presence/absence of MIAC in women with PTL and intact membranes. These metabolic changes might be indicative of enhanced glycolysis triggered by hypoxia conditions as a consequence of bacterial infection, thus explaining the utilization of alternative energy sources in an attempt to replenish glucose.

Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction.

T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.

Pathophysiologic processes have an impact on the plasma metabolomic signature of endometriosis patients.

OBJECTIVE: To evaluate potential variations in the plasma metabolomic profile of endometriosis patients as a consequence of pathophysiologic alterations associated with this disorder. DESIGN: Prospective study. For each subject, a plasma sample was collected after overnight fasting and before surgery. SETTING: University medical center. PATIENT(S): The clinical cohort included 50 endometriosis patients, diagnosed at early (n = 6) and advanced (n = 44) stages of the disease, and 23 healthy women. All volunteers underwent diagnostic laparoscopy to visually confirm the presence or absence of endometriotic lesions. INTERVENTION(S): Metabolomic profiling of plasma samples based on 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with statistical approaches. MAIN OUTCOME MEASURE(S): Comparative identification of metabolites present in plasma from endometriosis patients and healthy women. RESULT(S): The plasma metabolomic profile of endometriosis patients was characterized by increased concentration of valine, fucose, choline-containing metabolites, lysine/arginine, and lipoproteins and decreased concentration of creatinine compared with healthy women. Metabolic alterations identified in the plasma metabolomic profile of endometriosis patients correlate with pathophysiologic events previously described in the progression of this disease. CONCLUSION(S): The results highlight the potential of 1H-NMR-based metabolomics to characterize metabolic alterations associated with endometriosis in plasma samples. This information could be useful to get a better understanding of the molecular mechanisms involved in the pathogenesis of endometriosis, thus facilitating the noninvasive diagnosis of this pathology at early stages.

Nuclear magnetic resonance metabolomic profiling of urine provides a noninvasive alternative to the identification of biomarkers associated with endometriosis.

OBJECTIVE: To investigate whether urine metabolomic profile can be used to identify biomarkers associated to endometriosis. DESIGN: Prospective study. For each subject, a urine sample was collected after overnight fasting and before surgery. SETTING: University medical center. PATIENT(S): The clinical cohort included 45 endometriosis patients, diagnosed at early (n = 6) and advanced (n = 39) stages of the disease, and 36 healthy women. All women underwent diagnostic laparoscopy to visually confirm the presence or absence of endometriotic lesions. INTERVENTION(S): Metabolomic profiling of urine samples based on (1)H-nuclear magnetic resonance (NMR) spectroscopy in combination with statistical approaches. MAIN OUTCOME MEASURE(S): Comparative identification of metabolites present in urine from endometriosis patients and healthy women. RESULT(S): The urine metabolomic profile of endometriosis patients exhibited higher concentrations of N(1)-methyl-4-pyridone-5-carboxamide, guanidinosuccinate, creatinine, taurine, valine, and 2-hydroxyisovalerate and decreased concentrations of lysine compared with healthy women. Most of these metabolites are involved in inflammation and oxidative stress processes. These pathophysiologic events had been previously described to be present in ectopic endometrial proliferation foci. CONCLUSION(S): Overall, the results demonstrate the potential of (1)H-NMR-based metabolomics, a rapid and noninvasive approach, to identify metabolic changes associated to endometriosis in urine samples. This information could be useful to get a better understanding of the pathogenesis of endometriosis, thus providing support to the noninvasive diagnosis of this pathology.

Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex.

Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5'-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.