Levels Of Glycerophosphocholine Research Articles

Gpcpd1-GPC metabolic pathway is dysfunctional in aging and its deficiency severely perturbs glucose metabolism.

Skeletal muscle plays a central role in the regulation of systemic metabolism during lifespan. With aging, this function is perturbed, initiating multiple chronic diseases. Our knowledge of mechanisms responsible for this decline is limited. Glycerophosphocholine phosphodiesterase 1 (Gpcpd1) is a highly abundant muscle enzyme that hydrolyzes glycerophosphocholine (GPC). The physiological functions of Gpcpd1 remain largely unknown. Here we show, in mice, that the Gpcpd1-GPC metabolic pathway is perturbed in aged muscles. Further, muscle-specific, but not liver- or fat-specific, inactivation of Gpcpd1 resulted in severely impaired glucose metabolism. Western-type diets markedly worsened this condition. Mechanistically, Gpcpd1 muscle deficiency resulted in accumulation of GPC, causing an 'aged-like' transcriptomic signature and impaired insulin signaling in young Gpcpd1-deficient muscles. Finally, we report that the muscle GPC levels are markedly altered in both aged humans and patients with type 2 diabetes, displaying a high positive correlation between GPC levels and chronological age. Our findings reveal that the muscle GPCPD1-GPC metabolic pathway has an important role in the regulation of glucose homeostasis and that it is impaired during aging, which may contribute to glucose intolerance in aging.

The Lysophospholipase PNPLA7 Controls Hepatic Choline and Methionine Metabolism.

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.

The alteration of the expression level of neuropathy target esterase in human neuroblastoma SK-N-SH cells disrupts cellular phospholipids homeostasis

Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95–99% inhibition at 50 μM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels.

Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility.

Simple SummaryAlthough semen cryopreservation has facilitated the diffusion of artificial insemination and in vitro fertilization in cattle, it still represents a major factor affecting sperm fertility. It is known that cryopreservation induces the loss of fertility-associated proteins, while the effect on metabolites has not been evaluated, although several compounds affect sperm physiology and fertility. The aim of the present work was to study the metabolome in bovine sperm and seminal plasma after cryopreservation and to correlate the metabolic profile of high- and low-fertility bulls in order to identify fertility markers. The analysis, carried out by liquid chromatography–mass spectrometry methods, revealed differences in metabolite contents between fresh and cryopreserved semen, both at cellular and plasmatic levels. Interestingly, metabolites showing variation have important functions related to fertility. In addition, the study highlighted the differences in lipid profile between high- and low-fertility bulls. The identification of new potential fertility markers is of high economic impact. In addition, it opens the way for the development of corrective strategies to improve the fertility of low-fertility bulls.Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.

A serum metabolomics study of patients with nAMD in response to anti-VEGF therapy

Intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is the current standard of treatment for choroidal neovascularization (CNV) secondary to neovascular age-related macular degeneration (nAMD), but there are no diagnostic tools to predict response of these therapies. We hypothesize that differences in baseline metabolic profiles of patients with nAMD may influence responsiveness to anti-VEGF therapy, and thus provide prognosticating information for these patients. A prospective study was performed on 100 patients with nAMD treated with anti-VEGF therapy. We classified patients into two groups: responders (n = 54) and non-responders (n = 46). The expression levels of glycerophosphocholine,LysoPC (18:2) and PS (18:0/20:4) were higher in non-responders and these findings were verified in the validation cohort, implicating that reductions in these three metabolites can be used as predictors for responsiveness to anti-VEGF therapy during the initial loading phase for patients with nAMD. Our study also provided new insights into the pathophysiological changes and molecular mechanism of anti- VEGF therapy for nAMD patients.

Plasma Metabolomics Profiling in Maintenance Hemodialysis Patients Based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry

Background: Key pathogenetic mechanisms underlying renal disease progression are unaffected by current treatment. Metabolite profiling has significantly contributed to a deeper understanding of the biochemical metabolic networks and pathways in disease, but the biochemical details in maintenance hemodialysis (MHD) patients remain largely undefined. Methods: The metabolic fingerprinting of plasma samples from 19 MHD patients and 12 healthy controls was characterized using liquid chromatography quadrupole time-of-flight mass spectrometry. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to analyze the metabolic data. Results: The plasma metabolite profile distinguished the MHD patients from the healthy controls successfully by using both PCA and OPLS-DA models. Sixty-three metabolites were identified as the key metabolites to discriminate the MHD patients from healthy controls, involving several metabolic pathways (all p < 0.05). An increase in plasma levels of D-glucose, hippuric acid, androsterone glucuronide, indolelactic acid, and a reduction in plasma levels of glycerophosphocholine, serotonin, L-lactic acid, phytosphingosine, and several lysophosphatidylcholine were observed in MHD patients compared to healthy subjects. Metabolomics analysis combined with KEGG pathway enrichment analysis revealed that non-alcoholic fatty liver disease, choline metabolism in cancer, the forkhead box O signaling pathway, and the hypoxia-inducible factor-1 signaling pathway in MHD patients were significantly changed (p < 0.05). Conclusion: The identification of a novel signaling pathway and key metabolite markers in MHD patients provides insights into potential pathogenesis and valuable pharmacological targets for end-stage renal disease.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Elucidating the survival and response of carbapenem resistant Klebsiella pneumoniae after exposure to imipenem at sub-lethal concentrations

ABSTRACTThe increasing prevalence of antibiotic resistant pathogens poses a serious threat to global health. However, less emphasis has been placed to co-relate the gene expression and metabolism of antibiotic resistant pathogens. This study aims to elucidate gene expression and variations in metabolism of multidrug resistant Klebsiella pneumoniae after exposure to antibiotics. Phenotypic responses of three genotypically distinct carbapenem resistant Klebsiella pneumoniae (CRKP) strains untreated and treated with sub-lethal concentrations of imipenem were investigated via phenotype microarrays (PM). The gene expression and metabolism of the strain harboring blaNDM-1 before and after exposure to sub-lethal concentration of imipenem were further investigated by RNA-sequencing (RNA-Seq) and 1H NMR spectroscopy respectively. Most genes related to cell division, central carbon metabolism and nucleotide metabolism were downregulated after imipenem treatment. Similarly, 1H NMR spectra obtained from treated CRKP showed decrease in levels of bacterial end products (acetate, pyruvate, succinate, formate) and metabolites involved in nucleotide metabolism (uracil, xanthine, hypoxanthine) but elevated levels of glycerophosphocholine. The presence of anserine was also observed for the treated CRKP while FAPγ-adenine and methyladenine were only present in untreated bacterial cells. As a conclusion, the studied CRKP strain exhibited decrease in central carbon metabolism, cell division and nucleotide metabolism after exposure to sub-lethal concentrations of imipenem. The understanding of the complex biological system of this multidrug resistant bacterium may help in the development of novel strategies and potential targets for the management of the infections.

Abstract 2120: Distinct molecular effects of chemotherapeutic agents on choline phospholipid metabolism of triple-negative breast cancer cells

Abstract The magnetic resonance spectroscopy (MRS)-detected total choline (tCho) signal is a promising non-invasive surrogate marker able to predict chemotherapy response in breast cancer patients early on. However, the molecular mechanisms by which common chemotherapeutic drugs affect the tCho signal, which consists of glycerophosphocholine (GPC), phosphocholine (PC), and free choline (Cho), are mostly unknown. Here we have employed some widely used cancer chemotherapeutic drugs such as doxorubicin, paclitaxel, and vinorelbine to treat triple-negative human MDA-MB-231 breast cancer cells to elucidate their molecular effects on choline phospholipid metabolism. High-resolution (HR) 1H MRS of water-soluble cell extracts was employed to detect changes in cellular choline metabolite profiles, and quantitative RT-PCR (qRT-PCR) to assess the corresponding changes in the expression levels of choline-metabolizing enzymes. After 48 hours of doxorubicin treatment, the GPC levels in MDA-MB-231 cells significantly increased, while PC level decreased, and the tCho concentration remained unchanged. Vinorelbine treatment displayed a comparable effect to doxorubicin, but the GPC increase and PC decrease occurred to a lesser extent. Paclitaxel treatment caused an increased GPC level along with an unaltered PC level, leading to a slightly increased tCho level. In all these cases, the PC/GPC ratio decreased from 3.5 in the control to about 2.0 in paclitaxel- or vinorelbine-treated samples, to about 1.0 in doxorubicin-treated samples. qRT-PCR-detected mRNA expression levels showed that GDPD6 and Chkα were downregulated by doxorubicin. GDPD5, GDPD6, and Chkα genes displayed a reduced mRNA expression level following vinorelbine treatment, while no significant change in these three genes was detected after paclitaxel treatment. For comparison, the known GDPD6 inhibitor dipyridamole significantly increased cellular GPC levels, but, as expected, did not affect GDPD6 mRNA expression levels as it acts as an enzyme inhibitor. Our study demonstrates that choline containing metabolites change differently depending on the type of drug used for breast cancer treatment. However, all tested chemotherapeutic treatments resulted in metabolic alteration away from the ‘cholinic phenotype’, which was caused in some cases by decreases in expression of Chkα, GDPD6, and GDPD5 following drug treatment. The choline metabolite concentrations of GPC and/or PC and/or the PC/GPC ratio may serve as non-invasive surrogate makers of therapeutic response in triple-negative breast cancer patients undergoing chemotherapy. Note: This abstract was not presented at the meeting. Citation Format: Menglin Cheng, Zaver M. Bhujwalla, Kristine Glunde. Distinct molecular effects of chemotherapeutic agents on choline phospholipid metabolism of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2120. doi:10.1158/1538-7445.AM2017-2120

Skin melanocytes and fibroblasts show different changes in choline metabolism during cellular senescence

Unmodified cells undergo only a limited number of cell divisions until they enter a state termed cellular senescence. Other triggers like cytotoxic compounds can also induce cell senescence. Since cell senescence represents a major mechanism of tumor suppression this cellular state has attracted increasing attention. Different markers like senescence-associated β-galactosidase (SAβGal), senescence-associated heterochromatic foci (SAHF) or certain metabolic changes have been identified to be characteristic for senescent cells; however, data is often limited to fibroblasts − the cardinal model system for cellular senescence. In order to investigate whether metabolic changes during senescence are cell type independent, skin fibroblasts and skin melanocytes have been examined. Expression of the senescence marker p16 could be detected in skin fibroblasts but not in melanocytes of this specific donor, rendering the senescent phenotype not fully ascertained for the melanocytes. Metabolic profiles of senescent cells and controls have been determined using NMR spectroscopy. Changes in metabolism are different for fibroblasts and melanocytes. Senescent melanocytes showed lower levels of phosphocholine whereas for fibroblasts in accordance with literature, levels of glycerophosphocholine were increased during senescence. Although no general metabolic marker for cellular senescence exists, the same metabolic pathway seems to be affected for both cell types.

Abstract P6-02-07: Metabolomic analysis by nuclear magnetic resonance spectroscopy discriminates hormone receptor positive/HER2 negative breast cancer cell lines resistant to palbociclib

Abstract Introduction: Clinical trials of palbociclib in combination with endocrine therapy have recently shown unprecedented activity for the treatment of hormone receptor positive/HER2 negative (HR+/HER2neg) advanced breast cancer. However, de novo and acquired resistance to palbociclib limit its clinical utility. Moreover, combining palbociclib with endocrine therapy increases toxicity and costs of the treatment. Identifying patients more likely to benefit from this compound and understanding the mechanisms of resistance to palbociclib is critical. In this study we investigated whether metabolomic profiles of breast cancer cell lines with acquired resistance to palbociclib (PDR) differ from their sensitive counterpart (PDS). In addition we sought to identify metabolic biomarkers of sensitivity to palbociclib by analyzing a breast cancer cell line unable to acquire resistance to palbociclib. Material and methods: We have established in our lab three PDR HR+/HER2neg breast cancer models (MCF7L, T47D and ZR75-1) by chronically exposing cells to escalating doses of palbociclib. PDR derivatives show IC50 values 6 to 30 times higher than their PDS counterparts. One additional model, CAMA-1, was unable to develop resistance. Whole-cell lysates and conditioned cell culture media from five replicates of each of the PDS and PDR models and from CAMA-1 were analyzed by nuclear magnetic resonance (NMR). Principal component analysis (PCA) was used as first exploratory analysis and as dimension reduction technique. Canonical Analysis (CA) was used to discriminate different groups. Differentially expressed metabolites between PDR and PDS models and between CAMA-1 and PDS cells were analyzed. Results: Unsupervised PCA analyses of H NMR spectra, in which no information about PDS and PDR was inserted in the statistical model, correctly identified individual cell lines on both whole-cell lysates and conditioned media. However this analysis did not discriminate PDS from PDR within each model. Using a supervised approach, in which the statistical model was trained to discriminate between PDS and PDR, these groups were categorized with accuracy of 80% using whole-cell lysates and of 65% using conditioned media, using a cross-validation analysis by repeatedly testing the model on blind samples. CAMA-1 was correctly identified as a PDS model; however it showed a distinct metabolic profile compared to other PDS models. Over 30 metabolites were identified as differentially expressed between PDS and PDR models in lysates and conditioned media, but only glycerophosphocholine levels in conditioned media remained significantly higher in PDR compared to PDS models after correction for multiple testing. Conclusions: In this study we show that analysis of metabolic profile of cells lysates discriminates PDR from PDS cell lines with a high accuracy. Analysis of metabolic pathways implicated in resistance/sensitivity to palbociclib is ongoing and might help identifying new targets to overcome resistance. Additionally, metabolites associated with palbociclib resistance may be potentially tested in clinical samples as biomarkers for patients stratification. Further studies are warranted. Citation Format: Bonechi M, Guarducci C, Meoni G, Tenori L, Biagioni C, Schiff R, Osborne CK, Luchinat C, Di Leo A, Malorni L, Migliaccio I. Metabolomic analysis by nuclear magnetic resonance spectroscopy discriminates hormone receptor positive/HER2 negative breast cancer cell lines resistant to palbociclib [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-07.

Metabolite profiling of infection-associated metabolic markers of onchocerciasis

The global efforts for onchocerciasis elimination may require additional tools (safe micro and macrofilaricidal drugs, vaccines and biomarkers) as elimination efforts move toward the “end game”. Efforts toward the identification of suitable biomarkers have focused on specific protein(s) and/or nucleic acids, but metabolites present an alternative option as they have limited half-lives and are the result of combinatorial effects. In comparison to previously used methodology of LC–MS for metabolomic approaches, we used a non-targeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) to analyze the serum metabolic profiles of Ov-infected and −uninfected individuals (n=20). We identified 286 known metabolites (167 in the cation mode and 119 in the anion mode). In addition, putative metabolites were identified based on KEGG (51), HMDB (37) and HMT (6) databases. One hundred ten of these putative metabolites were quantified based on peak areas of internal standards and their ability to be mapped to known pathways (primary-, carbon-, lipid-, amino acid-, nucleotide and coenzyme-metabolism). Multivariate analysis demonstrated clustering and segregation of some of these metabolites to either the infected or control groups. The levels of serotonin, hypoxanthine, pipecolic acid and inosine were significantly elevated in those with onchocerciasis, whereas the levels of glycerophosphocholine, choline and adenine were significantly lower. This non-targeted metabolomic approach provides a global view of the metabolic variations that occur during Ov infection and thus allow the discovery of key metabolites (and associated pathways) that may serve as useful biomarkers in human onchocerciasis.

Pro-Oxidant Role of Silibinin in DMBA/TPA Induced Skin Cancer: 1H NMR Metabolomic and Biochemical Study.

Silibinin, a major bioactive flavonolignan in Silybum marianum, has received considerable attention in view of its anticarcinogenic activity. The present study examines its anticancer potential against 7, 12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin cancer. Male LACA mice were randomly segregated into 4 groups: Control, DMBA/TPA, Silibinin and Silibinin+DMBA/TPA. Tumors in DMBA/TPA and Silibinin+DMBA/TPA groups were histologically graded as squamous cell carcinoma. In the Silibinin+DMBA/TPA group, significant reduction in tumor incidence (23%), tumor volume (64.4%), and tumor burden (84.8%) was observed when compared to the DMBA/TPA group. The underlying protective mechanism of Silibinin action was studied at pre-initiation (2 weeks), post-initiation (10 weeks) and promotion (22 weeks) stages of the skin carcinogenesis. The antioxidant nature of Silibinin was evident at the end of 2 weeks of its treatment. However, towards the end of 10 and 22 weeks, elevated lipid peroxidation (LPO) levels indicate the pro-oxidative nature of Silibinin in the cancerous tissue. TUNEL assay revealed enhanced apoptosis in the Silibinin+DMBA/TPA group with respect to the DMBA/TPA group. Therefore, it may be suggested that raised LPO could be responsible for triggering apoptosis in the Silibinin+DMBA/TPA group. 1H Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the metabolic profile of the skin /skin tumors. Dimethylamine (DMA), glycerophosphocholine (GPC), glucose, lactic acid, taurine and guanine were identified as the major contributors for separation between the groups from the Principal Component Analysis (PCA) of the metabolite data. Enhanced DMA levels with no alteration in GPC, glucose and lactate levels reflect altered choline metabolism with no marked Warburg effect in skin tumors. However, elevated guanine levels with potent suppression of taurine and glucose levels in the Silibinin+DMBA/TPA group are suggestive of the pro-oxidative nature of Silibinin in regressing tumors. Thus, supporting the theory of augmented LPO levels resulting in increased apoptosis in the skin tumors treated with Silibinin.

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration, and invasion.

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC-specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF-7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA-MB-231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic clusters of breast cancer in relation to gene- and protein expression subtypes.

BackgroundThe heterogeneous biology of breast cancer leads to high diversity in prognosis and response to treatment, even for patients with similar clinical diagnosis, histology, and stage of disease. Identifying mechanisms contributing to this heterogeneity may reveal new cancer targets or clinically relevant subgroups for treatment stratification. In this study, we have merged metabolite, protein, and gene expression data from breast cancer patients to examine the heterogeneity at a molecular level.MethodsThe study included primary tumor samples from 228 non-treated breast cancer patients. High-resolution magic-angle spinning magnetic resonance spectroscopy (HR MAS MRS) was performed to extract the tumors metabolic profiles further used for hierarchical cluster analysis resulting in three significantly different metabolic clusters (Mc1, Mc2, and Mc3). The clusters were further combined with gene and protein expression data.ResultsOur result revealed distinct differences in the metabolic profile of the three metabolic clusters. Among the most interesting differences, Mc1 had the highest levels of glycerophosphocholine (GPC) and phosphocholine (PCho), Mc2 had the highest levels of glucose, and Mc3 had the highest levels of lactate and alanine. Integrated pathway analysis of metabolite and gene expression data uncovered differences in glycolysis/gluconeogenesis and glycerophospholipid metabolism between the clusters. All three clusters had significant differences in the distribution of protein subtypes classified by the expression of breast cancer-related proteins. Genes related to collagens and extracellular matrix were downregulated in Mc1 and consequently upregulated in Mc2 and Mc3, underpinning the differences in protein subtypes within the metabolic clusters. Genetic subtypes were evenly distributed among the three metabolic clusters and could therefore contribute to additional explanation of breast cancer heterogeneity.ConclusionsThree naturally occurring metabolic clusters of breast cancer were detected among primary tumors from non-treated breast cancer patients. The clusters expressed differences in breast cancer-related protein as well as genes related to extracellular matrix and metabolic pathways known to be aberrant in cancer. Analyses of metabolic activity combined with gene and protein expression provide new information about the heterogeneity of breast tumors and, importantly, the metabolic differences infer that the clusters may be susceptible to different metabolically targeted drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-016-0152-x) contains supplementary material, which is available to authorized users.

Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

Background:Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management.Methods:Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance.Results:Comparison of NMR metabolite profiles obtained from control (CALS) and EGFR TKI-resistant (CALR) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CALR relative to CALS monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism.Conclusions:Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Short call abstracts.

Short call abstracts.

IDH1 R132H Mutation Generates a Distinct Phospholipid Metabolite Profile in Glioma

Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To investigate this hypothesis, we performed (31)P-MRS imaging in mouse xenograft models of four human gliomas, one of which harbored the IDH1-R132H mutation. (31)P-MR spectra from the IDH1-mutant tumor displayed a pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoethanolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by (31)P high-resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was established by in vitro (31)P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phosphoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma.

Phosphorus Magnetic Resonance Spectroscopic Imaging at 7 T in Patients With Prostate Cancer

The aim of this study was to identify characteristics of phosphorus (P) spectra of the human prostate and to investigate changes of individual phospholipid metabolites in prostate cancer through in vivo P magnetic resonance spectroscopic imaging (MRSI) at 7 T. In this institutional review board-approved study, 15 patients with biopsy-proven prostate cancer underwent T2-weighted magnetic resonance imaging and 3-dimensional P MRSI at 7 T. Voxels were selected at the tumor location, in normal-appearing peripheral zone tissue, normal-appearing transition zone tissue, and in the base of the prostate close to the seminal vesicles. Phosphorus metabolite ratios were determined and compared between tissue types. Signals of phosphoethanolamine (PE) and phosphocholine (PC) were present and well resolved in most P spectra in the prostate. Glycerophosphocholine signals were observable in 43% of the voxels in malignant tissue, but in only 10% of the voxels in normal-appearing tissue away from the seminal vesicles. In many spectra, independent of tissue type, 2 peaks resonated in the chemical shift range of inorganic phosphate, possibly representing 2 separate pH compartments. The PC/PE ratio in the seminal vesicles was highly elevated compared with the prostate in 5 patients. A considerable overlap of P metabolite ratios was found between prostate cancer and normal-appearing prostate tissue, preventing direct discrimination of these tissues. The only 2 patients with high Gleason scores tumors (≥4+5) presented with high PC and glycerophosphocholine levels in their cancer lesions. Phosphorus MRSI at 7 T shows distinct features of phospholipid metabolites in the prostate gland and its surrounding structures. In this exploratory study, no differences in P metabolite ratios were observed between prostate cancer and normal-appearing prostate tissue possibly because of the partial volume effects of small tumor foci in large MRSI voxels.

Silencing of the glycerophosphocholine phosphodiesterase GDPD5 alters the phospholipid metabolite profile in a breast cancer model in vivo as monitored by (31) P MRS.

Abnormal choline phospholipid metabolism is an emerging hallmark of cancer, which is implicated in carcinogenesis and tumor progression. The malignant metabolic phenotype is characterized by high levels of phosphocholine (PC) and relatively low levels of glycerophosphocholine (GPC) in aggressive breast cancer cells. Phosphorus ((31) P) MRS is able to non-invasively detect these water-soluble metabolites of choline as well as ethanolamine phospholipid metabolism. Here we have investigated the effects of stably silencing glycerophosphoester diesterase domain containing 5 (GDPD5), which is an enzyme with glycerophosphocholine phosphodiesterase activity, in MDA-MB-231 breast cancer cells and orthotopic tumor xenografts. Tumors in which GDPD5 was stably silenced with GDPD5-specific shRNA contained increased levels of GPC and phosphoethanolamine (PE) compared with control tumors.