The study of RAS-induced metabolic reprogramming and the role of the pentose phosphate pathway in tumor metabolism

[spa] La presente tesis doctoral se centra en las adaptaciones metabólicas inducidas por la activación de oncogenes así como en el potencial del entramado metabólico como diana antitumoral. A lo largo de los últimos años, ha resurgido un renovado interés en el estudio del metabolismo, particularmente en el metabolismo de las células tumorales, dando lugar a una nueva disciplina conocida como metabolismo tumoral. Numerosas investigaciones se han centrado en la asociación entre mutaciones en oncogenes o genes supresores de tumores con perfiles metabólicos característicos, en busca de dependencias metabólicas que ofrezcan nuevas posibilidades para el tratamiento de los tumores. La búsqueda de alteraciones metabólicas que constituyan vulnerabilidades de la célula tumoral representa la piedra angular de esta interesante disciplina. Así, esta tesis doctoral tiene como objetivo general elucidar las alteraciones metabólicas que acompañan a la mutación de oncogenes y explorar el potencial del entramado metabólico como diana antitumoral. Por tanto, los objetivos principales de este trabajo son los siguientes: i) análisis de la reprogramación metabólica inducida por la activación oncogénica de RAS empleando líneas celulares transfectadas de manera estable con copias mutadas de los oncogenes K-RAS y H-RAS y, ii) validación de la vía de las pentosas fosfato como potencial diana antitumoral y estudio de su papel en el metabolismo tumoral de modelos celulares de cáncer de colon y de mama. Así, en este trabajo de tesis doctoral hemos concluido que la activación oncogénica de RAS promueve una profunda reprogramación del metabolismo induciendo cambios significativos en la glucólisis, la vía de las pentosas fosfato, el metabolismo de la glutamina y la lipogénesis. Por otro lado, hemos determinado que la inhibición de la vía de las pentosas fosfato tiene distintos efectos según el tipo de tumor. La inhibición de la G6PD en la línea celular de cáncer colon HT29 no produjo efectos sobre la proliferación mientras que su inhibición en células de cáncer de mama MCF7 indujo una notable reducción de la proliferación y un incremento de la muerte celular. Por otra parte, en la inhibición en MCF7 del otro enzima clave de la vía de las pentosas fosfato, la TKT, no se observaron cambios significativos en términos de proliferación y viabilidad celular. Además, en este trabajo también se ha puesto de manifiesto una conexión funcional entre la vía de las pentosas fosfato y el metabolismo de la glutamina en ambos modelos celulares, sugiriendo un papel complementario de estas dos vías metabólicas.[eng] The present doctoral thesis is focused on the metabolic adaptations induced by oncogene activation as well as the potential role of the metabolic network as antitumor therapy. Over the last years, it has emerged a renewed interest in the field of metabolism, particularly in cancer metabolism. Great efforts have been focused on the association of mutated oncogenes or tumor suppressor genes and tumor metabolic profiles, in the search of metabolic dependencies that offer new potential avenues for cancer treatment. The pursuit of discovering tumor metabolic alterations in which cancer cells rely on has represented the cornerstone of this interesting discipline. Thus, this thesis is part of this recent and promising scientific current and is intended to shed light on the metabolic alterations accompanying oncogene mutation and on potential metabolic pathways that might be of therapeutic interest in the future. Hence, the objectives of this thesis can be divided into two specific aims: i) analysis of the metabolic reprogramming of RAS oncogenic activation using stable transfected cell lines with mutated copies of K-RAS and H-RAS and ii) validation of the pentose phosphate pathway as a potential therapeutic target and exploration of its role within tumor metabolism in colon and breast cancer cell models. Thus, according to the proposed objectives, the main conclusions obtained are as follow: 1. The study of flux distribution in combination with metabolic control analysis performed by analyzing solely the sign of fixed-sign control coefficients, is a reliable approach to identify the key enzymes involved in metabolic reprogramming. The use of this methodology has allowed us to identify an increase in glycolysis and PPP fluxes as metabolic features of KRAS-induced metabolic reprogramming and to propose G6PD, PK and LDH as the key enzymes responsible for this metabolic transition. 2. H-RAS oncogenic activation reprograms glucose and glutamine metabolism by enhancing glycolytic and PPP fluxes as well as mitochondrial metabolism. Glutamine is responsible for sustaining the activated mitochondrial metabolism in BJ-HRasV12, while glucose-derived carbons in the mitochondria are primarily used to fuel lipogenesis. Moreover, lipogenesis is overactivated in BJ-HRasV12 cells, which are more sensitive to FAS inhibition than BJ cells. 3. G6PD enzyme is overactivated in colon cancer cells with oncogenic activation of the RAS signaling pathway. Nevertheless, G6PD seems to be dispensable for proliferation and survival in BRAF-mutated HT29 cell line. Furthermore, a new connection between PPP and glutamine metabolism has been unveiled, as G6PD is overexpressed in HT29 cells under glutamine-deprived conditions by a mechanism involving a concomitantly increase in ROS levels and NRF2 induction. 4. G6PD enzyme is important in proliferation, survival and regulation of ROS levels in breast cancer MCF7 cells. However, it exerts a low regulation over ribose synthesis flux through the oxidative branch of PPP. G6PD inhibition enhances glycolytic flux, promotes lactate secretion and increases glutamine consumption, which is used to maintain energy homeostasis, although it is not essential for cell proliferation. 5. TKT enzyme is dispensable for proliferation of breast cancer MCF7 cells, but it exerts a high control over ribose synthesis flux through the nonoxidative branch of PPP. TKT impairment reduces glycolytic flux and increases the consumption of glutamine, which is intended to maintain energy homeostasis but it is not essential for cell proliferatio

Oxidative pentose phosphate pathway enzyme 6-phosphogluconate dehydrogenase plays a key role in breast cancer metabolism

The pentose phosphate pathway (PPP) plays an essential role in the metabolism of breast cancer cells for the management of oxidative stress and the synthesis of nucleotides. 6-phosphogluconate dehydrogenase (6PGD) is one of the key enzymes of the oxidative branch of PPP and is involved in nucleotide biosynthesis and redox maintenance status. Here, we aimed to analyze the functional importance of 6PGD in a breast cancer cell model. Inhibition of 6PGD in MCF7 reduced cell proliferation and showed a significant decrease in glucose consumption and an increase in glutamine consumption, resulting in an important alteration in the metabolism of these cells. No difference in reactive oxygen species (ROS) production levels was observed after 6PGD inhibition, indicating that 6PGD, in contrast to glucose 6-phosphate dehydrogenase, is not involved in redox balance. We found that 6PGD inhibition also altered the stem cell characteristics and mammosphere formation capabilities of MCF7 cells, opening new avenues to prevent cancer recurrance after surgery or chemotherapy. Moreover, inhibition of 6PGD via chemical inhibitor S3 resulted in an induction of senescence, which, together with the cell cycle arrest and apoptosis induction, might be orchestrated by p53 activation. Therefore, we postulate 6PGD as a novel therapeutic target to treat breast cancer

MIDcor, an R-program for deciphering mass interferences in mass spectra of metabolites enriched in stable isotopes

Background: Tracing stable isotopes, such as 13 C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study. Results: Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D)between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2)D was found to decrease when the tested substance was 13 C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13 C labeling is known a priori. Conclusion: Using the developed program improves the reliability of stable isotope tracer data analysis

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Glutamine modulates expression and function of glucose 6-phosphate dehydrogenase via NRF2 in colon cancer cells.

Nucleotide pools need to be constantly replenished in cancer cells to support cell proliferation. The synthesis of nucleotides requires glutamine and 5-phosphoribosyl-1-pyrophosphate produced from ribose-5-phosphate via the oxidative branch of the pentose phosphate pathway (ox-PPP). Both PPP and glutamine also play a key role in maintaining the redox status of cancer cells. Enhanced glutamine metabolism and increased glucose 6-phosphate dehydrogenase (G6PD) expression have been related to a malignant phenotype in tumors. However, the association between G6PD overexpression and glutamine consumption in cancer cell proliferation is still incompletely understood. In this study, we demonstrated that both inhibition of G6PD and glutamine deprivation decrease the proliferation of colon cancer cells and induce cell cycle arrest and apoptosis. Moreover, we unveiled that glutamine deprivation induce an increase of G6PD expression that is mediated through the activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2). This crosstalk between G6PD and glutamine points out the potential of combined therapies targeting oxidative PPP enzymes and glutamine catabolism to combat colon cancer

MIDcor, an R-program for deciphering mass interferences in mass spectra of metabolites enriched in stable isotopes

Background: Tracing stable isotopes, such as 13 C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study. Results: Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D)between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2)D was found to decrease when the tested substance was 13 C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13 C labeling is known a priori. Conclusion: Using the developed program improves the reliability of stable isotope tracer data analysis

Global patterns of tree density are contingent upon local determinants in the world's natural forests.

Previous attempts to quantify tree abundance at global scale have largely neglected the role of local competition in modulating the influence of climate and soils on tree density. Here, we evaluated whether mean tree size in the world's natural forests alters the effect of global productivity on tree density. In doing so, we gathered a vast set of forest inventories including >3000 sampling plots from 23 well-conserved areas worldwide to encompass (as much as possible) the main forest biomes on Earth. We evidence that latitudinal productivity patterns of tree density become evident as large trees become dominant. Global estimates of tree abundance should, therefore, consider dependencies of latitudinal sources of variability on local biotic influences to avoid underestimating the number of trees on Earth and to properly evaluate the functional and social consequences

Prosthetic Valve Candida spp. Endocarditis: New Insights Into Long-term Prognosis—The ESCAPE Study

International audienceBackground: Prosthetic valve endocarditis caused by Candida spp. (PVE-C) is rare and devastating, with international guidelines based on expert recommendations supporting the combination of surgery and subsequent azole treatment.Methods: We retrospectively analyzed PVE-C cases collected in Spain and France between 2001 and 2015, with a focus on management and outcome.Results: Forty-six cases were followed up for a median of 9 months. Twenty-two patients (48%) had a history of endocarditis, 30 cases (65%) were nosocomial or healthcare related, and 9 (20%) patients were intravenous drug users. "Induction" therapy consisted mainly of liposomal amphotericin B (L-amB)-based (n = 21) or echinocandin-based therapy (n = 13). Overall, 19 patients (41%) were operated on. Patients <66 years old and without cardiac failure were more likely to undergo cardiac surgery (adjusted odds ratios [aORs], 6.80 [95% confidence interval [CI], 1.59-29.13] and 10.92 [1.15-104.06], respectively). Surgery was not associated with better survival rates at 6 months. Patients who received L-amB alone had a better 6-month survival rate than those who received an echinocandin alone (aOR, 13.52; 95% CI, 1.03-838.10). "Maintenance" fluconazole therapy, prescribed in 21 patients for a median duration of 13 months (range, 2-84 months), led to minor adverse effects.Conclusion: L-amB induction treatment improves survival in patients with PVE-C. Medical treatment followed by long-term maintenance fluconazole may be the best treatment option for frail patients

Role of age and comorbidities in mortality of patients with infective endocarditis.

The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups: A total of 3120 patients with IE (1327  There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in th