H-NMR Spectroscopy-Based Metabolomic Analysis of Human Liver Tissues Reveals Metabolic Biomarkers of Hepato-Cellular Carcinoma Developed in Cirrhotic or Non- Cirrhotic Disease

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NMR metabolomics of fibroblasts with inherited mitochondrial Complex I mutation reveals treatment-reversible lipid and amino acid metabolism alterations

International audienceIntroduction Elucidating molecular alterations due to mitochondrial Complex I (CI) mutations may help to understand CI deficiency (CID), not only in mitochondriopathies but also as it is caused by drugs or associated to many diseases. Objectives CID metabolic expression was investigated in Leber's hereditary optic neuropathy (LHON) caused by an inherited mutation of CI. Methods NMR-based metabolomics analysis was performed in intact skin fibroblasts from LHON patients. It used several datasets: one-dimensional H-1-NMR spectra, two-dimensional H-1-NMR spectra and quantified metabolites. Spectra were analysed using orthogonal partial least squares-discriminant analysis (OPLS-DA), and quantified metabolites using univariate statistics. The response to idebenone (IDE) and resveratrol (RSV), two agents improving CI activity and mitochondrial functions was evaluated. Results LHON fibroblasts had decreased CI activity (-43%, p 1 in OPLS-DA, p 1) and alanine (VIP > 1, p 1 and/or p < 0.05). Conclusion LHON fibroblasts display lipid and amino acid metabolism alterations that are reversed by mitochondria-targeted treatments, and can be related to adaptive changes. Findings bring insights into molecular changes induced by CI mutation and, beyond, CID of other origins

Etude mécanistique et fonctionnelle de la réponse du mélanome au traitement par chloroéthylnitrosourée, seul ou associé à une carence en méthionine

La méthionine (Met) est un acide aminé essentiel impliqué dans plusieurs voies métaboliques telles que la synthèse des protéines, polyamines, acides aminés soufrés (taurine et glutathion) et la méthylation de nombreuses espèces (créatine, phosphatidylcholine, ADN, protéines) par l'intermédiaire de sa forme active : la S-adénosylméthionine (SAM). Les cellules tumorales sont caractérisées par des altérations du métabolisme de la Met et témoignent d'une dépendance plus ou moins marquée à cet acide aminé. Certaines chimiothérapies ciblent le métabolisme de la Met dont les chloroéthylnitrosourées (CENU) qui, en plus de leur effet génotoxique, induisent une hypométhylation de l'ADN et ciblent le métabolisme de la Met. C'est la raison pour laquelle l'utilisation de régimes nutritionnels carencés en Met, seuls ou associés à des chimiothérapies, a ét étudiée pour améliorer l'efficacité du traitement de certains types de cancers (prostate, gliome, mélanome). Quelques travaux renseignent, au niveau transcriptionnel, sur l'effet de la carence en Met dans les tumeurs. Cependant, la réponse métabolique des tumeurs à une carence en Met seule ou associée à une chimiothérapie, et les voies de signalisation impliquées dans cette réponse sont mal connues. L'objectif de ce travail a été de définir les phénotypes métaboliques de dépendance et d'indépendance à la Met dans les tumeurs de mélanome B16 murin soumises à une carence nutritionnelle en Met, in vivo en modèle syngénique et in vitro en modèle de culture cellulaire. Nos travaux ont montré que la dépendance à la Met des cellules tumorales est caractérisée par une méthylation préférentielle des récepteurs de méthyles cytoplasmiques (créatine, phosphatidylcholine) et que l'indépendance à la Met est en revanche caractérisée par la méthylation préférentielle de macromolécules (ADN et protéines). Nous avons ensuite déterminé la réponse du métabolisme tumoral du mélanome B16 in vivo à un stress nutritionnel en Met, associé ou non à un traitement par CENU. Nos résultats démontrent que la carence en Met seule a des effets ubiquitaires partiellement réversibles après retour à une alimentation normale. L'association de la carence en Met au traitement par CENU potentialise l'inhibition de prolifération tumorale induite par la chimiothérapie. Ces travaux nous ont permis d'établir que la synergie thérapeutique observée à long terme est reliée à des désordres des métabolismes tumoraux énergétique (inhibition de la glutaminolyse) et phospholipidique (augmentation de la synthèse par méthylation de la phosphatidylcholine et de son hydrolyse). Ces travaux sont validés par des résultats obtenus in vitro en culture de cellules de mélanome B16. Sur la base des données concernant les effets du traitement par CENU et sur la réparttion des méthyles, nous avons analysé l'effet du traitement par CENU sur la méthylation dépendante de la SAM et l'activation de la protéine phosphatase 2A (PP2A). PP2A est impliquée dans la régulation de l'activation de Akt et de la stabilité de c-Myc et est également proposée comme un suppresseur de tumeur. Nos résultats montrent que le traitement par CENU induit l'augmentation de la méthylation et de l'activité de PP2A, ayant pour conséquence l'inactivation d'Akt et la dégradation de c-Myc. Ces résultats sont les 1ers à suggérer une intercation entre PP2A et PTEN, protéine suppresseur de tumeur fréquemment muté dans le mélanome. En conclusion, la présente thèse ouvre de nouvelles perspectives dans la compréhension des mécanismes métaboliques de la réponse à un stress nutritionnel, associé ou non à une chimiothérapie conventionnelle. Le rôle majeur des transméthylations dépendantes de la SAM dans la réponse au traitement par CENU a été démontré par l'analyse de la méthylation de PP2A. Des études futures viseront notamment à relier, par l'intermédiaire du métabolisme des acides gras et de la voie des pentoses phosphates, la régulation des métabolismes phospholipidique et énergétique à la régulation de la méthylation de PP2A. Ces données permettront d'aborder les mécanismes reliant la méthylation de PP2A et la carcinogenèse et participeront à la mise au point de thérapies ciblées efficaces.Methionine (Met) is an essential amino acid involved in several metabolic pathways such as protein synthesis, polyamines, sulfur amino acids (taurine and glutathione) production and transmethylation of many compounds (creatine and phosphatidylcholine synthesis, DNA, protein) through its active form : S-adenosylmethionine (SAM). Tumor Met metabolism is frequently altered and tumors are more or less dependent to this amino acid. Several anticancer treatments, such as chloroethylnitrosoureas (CENUs), besides their genotoxic effect, target Met metabolism and induce DNA hypomethylation. This is why the use of Met depleted regimen, alone or associated with chemotherapy, was studied to improve the efficiency of somme cancer treatments (prostate, glioma, melanoma). Few studies showed the effect of Met deprivation stress (MDS) in tumors at the transcriptional level. However, the metabolic response of tumors to MDS, alone or associated with chemotherapy, and signalling pathways involved in this response remain poorly known. The goals of our work were to define the metabolic phenotypes of Met dependence and independence in murine B16 melanoma tumors subjected to MDS, in vivo (syngeneic model) and in vitro (cultured cells). We showed that tumor Met dependency was characterized by preferential methylation of cytoplasmic methyl acceptors (creatine and phosphatidylcholine synthesis) and that Met independency was characterized by preferential methylation of macromolecules (DNA and proteins). Then we determined the response of B16 melanoma tumor metabolism to MDS in vivo, alone or in association with CENU treatment. Our results showed that Met deficiency lead to ubiquitous effects which were partially reversible, after return to a normal diet. Moreover association of Met deprivation to CENU treatment potentiated tumor growth inhibition induced by chemotherapy alone. This work enabled us to establish that the therapeutic synergistic effect observed was related to defects of the energetic (reduction of glutaminolysis) and phospholipid (increase of phosphatidylcholine synthesis by methylation and hydrolysis) metabolism. These results were confirmed by in vitro analyses in cultured B16 melanoma cells. On the basis of data related to the effects of CENU treatment on methyl groups distribution, we analysed the effect of CENU treatment on SAM dependent methylation and on protein phosphatase 2A (PP2A) activation. PP2A is involved in the regulation of Akt activation and in c-Myc stability and is also proposed as a tumor suppressor. PP2A shares its regulating role of Akt pathway with another tumor suppressor frequently mutated in melanoma, PTEN. Our results showed that CENU treatment induced an increase of PP2A methylation and activity. This led to Akt inactivation, c-Myc degradation and PTEN activation. Our results were the first to describe an interaction between PP2A and PTEN. In conclusion, the present doctoral thesis gave new prospects in the understanding of metabolic mechanisms involved in tumor response to a nutritional stress, alone or in association with conventional anticancer chemotherapy. The major role of SAM dependent transmethylations in the response to CENU treatment was highlighted by the analysis of PP2A methylation. The future prospects will particularly aim at connecting, the metabolic response of tumor cells to PP2A activation, especially threw phospholipid and energetic (glycolysis and pentose phosphate pathway) metabolism. These data give new informations linking PP2A methylation anticancer therapy, and give substantial informations for the development of effective targeted therapies.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Metabolomics and transcriptomics demonstrate severe oxidative stress in both localized chemotherapy-treated and bystander tumors

International audienceBackground: Localized radiotherapy is long known to cause damages to not only targeted but also non-targeted cells, the so-called bystander (BS) effect. Recently, BS effect was demonstrated in response to chemotherapy. To get further insight into the mechanism of chemotherapy-induced BS effect in vivo, we investigated the response of normal tissues and untreated BS melanomas, at distance from localized chemotherapy-treated melanomas. Methods: 816 melanoma cells were inoculated sc in one flank, in mice. Chemotherapy was administered intratumorally. After 3 weeks, untreated melanomas were implanted into the other flank. Tumors were analyzed morphologically, and using metabolomics and transcriptomics. Results: Locally-treated melanomas showed growth inhibition and pleiotropic metabolic and transcriptional alterations. Tumors recovered slow proliferation while exhibiting prominent oxidative stress response (decreased glutathione level, and increased expression of genes including Mt1, Gpx3, Sod3, and Hmox1). Plasma contained increased levels of oxidative stress products. However, liver and soleus muscle displayed unaltered morphological characteristics. In contrast, untreated BS melanomas induced from naive B16 cells showed reduced growth, marked oxidative stress response (decreased glutathione level, and increased expression of genes including Sod2, Gpx1 and Gsr), and ras oncogene expression alterations. Furthermore, metabolomics and transcriptomics enabled to estimate the proportion of cells undergoing the BS effect within treated tumors. Conclusion: Treatment of tumors with chemotherapy induces BS effects, underpinned by oxidative stress, in abnormal proliferating tissues in vivo, not in normal tissue, that significantly contribute to overall tumor response. General significance BS effect significantly contributes to response to chemotherapy, and may be exploited to improve overall response to cancer treatment

Metabolic Transformation of Normal Human Melanocytes Under Extracellular Stimuli

International audiencePrevious studies have suggested a link between cancer types and pathophysiological conditions associated to hyperinsulinemia such as type 2 diabetes, metabolic syndrome, obesity and chronic inflammation. In this report we investigated the link between hyperinsulinemia and cancer. As end points of transformation, the following criteria were used, including cell proliferation rate, soft agar colony formation, DNA content and methylation status, karyotype, oncoprotein transcription, expression and activity (PP2A, Akt/PAkt, cMyc, PTEN, PP1 and Ras). Previously, we and others found that PP2A activity controlled Akt and cMyc expression was decreased during carcinogenesis. Normal melanocyte cultures were exposed to high insulin and glucose concentrations added for 3 weeks. After 4 to 5 weeks, melanocytes exhibited shortened doubling time (2.7 days vs 5.6 days, Treated vs Control, P < 0.01). After three weeks treatment followed by 1 or 2 week(s) of culture in standard medium, treated melanocytes only were able to grow in soft agar colonies. Methylation of the catalytic subunit of PP2A was decreased (-40%, P < 0.001) and PP2A phosphatase activity reduced. Oncoprotein expression was altered with increased expression of PAkt (+35%, P < 0.01), Akt (+25%, P < 0.01) and c-Myc (+ 40%, P < 0.001), a weak increase of PP1 expression (+ 10%). In conclusion chronic exposure to insulin and glucose appear promotes normal human melanocyte transformation. It is probable that increased metabolism such as seen in inflammation plays a key role in carcinogenesi

Analysis method of omics data for metabolic interactions modeling in predictive carcinogenesis

SESSION 1 - EPIDEMIOLOGY - NUTRITIONAL PREVENTION AND CANCER RISKBackground:Currently research focus on the identification of precoce biomarkers is important in the field of Health especially in carcinogenesis. Indeed, specific biomarkers could be crucial to predict the first steps of cancer appearance and to improve cancer prevention and care. In that goal, omic approaches are massively used generating a large amount of data. Our understanding of these data flows become difficult, so it is necessary to develop adaptive tools in order to treat and apprehend them. In many omic studies on cancer-metabolism profiling, discriminant compounds are implicated in energetic pathways such as lipids, glutamin and creatin. For example, lipids are largely used for cancer cell membranes building and undergo a lesser energetic oxidation. Thus, lipids could be used to help for detection of precancer steps.Objective and Methods:This study aims to discern the role of metabolic biomarkers of interest in the first step ofcarcinogenesis. After identification of biomolecules from MS and NMR metabolomic data, we will modelize using softwares allowing dynamic view such as Metaboflux which analyzes flux distribution in metabolic networks, Cytoscape, an open source software, or MetExplore a web server linking metabolomic experiments and genome-scale metabolic networks. We will compare the limits of each software in order to implement the most appropriate to modelize metabolic interactions in predictive carcinogenesis. In this aim, three file formats i.e. Systems Biology Markup Language, JavaScript Object Notation and MAT a format for MATLAB, will be tested.Conclusion:Despite of the existing softwares, most of them do not allow a dynamic visualization of metabolic pathways. That’s why our study plans to implement the most appropriate tool in order to optimize the dynamic modeling of metabolic interaction

Human Hepatocellular Carcinoma Metabolomic Profile in Non Alcoholic Fatty Liver Disease (NAFLD) According to Severity of Fibrosis

Human Hepatocellular Carcinoma Metabolomic Profile in Non Alcoholic Fatty Liver Disease (NAFLD) According to Severity of Fibrosis. Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meetin

Metabolomic analysis by 1H-nuclear magnetic resonance spectroscopy suggest that there are 2 phenotypes of human hepatocellular carcinoma developed in non-alcoholic fatty liver disease according to fibrosis level (F0F1 vs F3F4)

POSTER PRESENTATIONS: Posters Thursday, 11 April 2019Liver tumours: Experimental and pathophysiologyMetabolomic analysis by 1H-nuclear magnetic resonance spectroscopy suggest that there are 2 phenotypes of human hepatocellular carcinoma developed in non-alcoholic fatty liver disease according to fibrosis level (F0F1 vs F3F4). International Liver Congres

Optimal Methionine-Free Diet Duration for Nitrourea Treatment: A Phase I Clinical Trial

In animal models, methionine (MET) restriction in association with chloroethylnitrosoureas led to a substantial improvement. On this basis, we initiated a Phase I clinical trial of dietary MET restriction in association with chloroethylnitrosourea (cystemustine) treatment for patients with recurrent glioma or metastatic melanoma. Our purpose was 1) to determine the optimal METfree diet duration for a maximum depletion of plasma MET an

L’étude des altérations métaboliques par spectroscopie RMN du carcinome hépato-cellulaire développé sur cirrhose ou sur foie non cirrhotique a mis en évidence l’existence de deux formes de CHC

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