Activation Of Metabolic Pathways Research Articles

Comprehensive analysis of metabolic pathway activity subtypes derived prognostic signature in hepatocellular carcinoma.

Metabolic reprogramming is one of the hallmarks of cancer, but metabolic pathway activity-related subtypes of hepatocellular carcinoma (HCC) have not been identified. Based on the quantification results of 41 metabolic pathway activities by gene set variation analysis, the training cohort (n=609, merged by TCGA and GSE14520) was clustered into three subtypes (C1, C2, and C3) with the nonnegative matrix factorization method. Totally 1371 differentially expressed genes among C1, C2, and C3 were identified, and an 8-gene risk score was established by univariable Cox regression analysis, least absolute shrinkage and selection operator method, and multivariable Cox regression analysis. C1 had the strongest metabolic activity, good prognosis, the highest CTNNB1 mutation rate, with massive infiltration of eosinophils and natural killer cells. C2 had the weakest metabolic activity, poor prognosis, was younger, was inclined to vascular invasion and advanced stage, had the highest TP53 mutation rate, exhibited a higher expression level of immune checkpoints, accompanied by massive infiltration of regulatory T cells. C3 had moderate metabolic activity and prognosis, the highest LRP1B mutation rate, and a higher infiltration level of neutrophils and macrophages. Internal cohorts (TCGA, n=370; GSE14520, n=239), external cohorts (ICGC, n=231; GSE116174, n=64), and clinical subgroup validation showed that the risk score was applicable for patients with diverse clinical features and was effective in predicting the prognosis and malignant progression of patients with HCC. Compared with the low-risk group, the high-risk group had a poor prognosis, enhanced cancer stem cell characteristics, activated DNA damage repair, weakened metabolic activity, cytolytic activity, and interferon response. We identified HCC subtypes from the perspective of metabolism-related pathway activity and proposed a robust prognostic signature for HCC.

Crosstalk of Epigenetic and Metabolic Signaling Underpinning Glioblastoma Pathogenesis.

Simple SummaryEpigenetic mechanisms can modulate key genes involved in the cellular metabolism of glioblastomas and participate in their pathogenesis by increasing their heterogeneity, plasticity, and malignancy. Although most epigenetic modifications can primarily promote the activity of metabolic pathways, they may also exert an inhibitory role. The detection of key metabolic alterations in gliomas regulated by epigenetic mechanisms will enable drug development and effective molecular targeting, improvement of therapeutic schemes, and patients’ management.Metabolic alterations in neoplastic cells have recently gained increasing attention as a main topic of research, playing a crucial regulatory role in the development and progression of tumors. The interplay between epigenetic modifications and metabolic pathways in glioblastoma cells has emerged as a key pathogenic area with great potential for targeted therapy. Epigenetic mechanisms have been demonstrated to affect main metabolic pathways, such as glycolysis, pentose phosphate pathway, gluconeogenesis, oxidative phosphorylation, TCA cycle, lipid, and glutamine metabolism by modifying key regulatory genes. Although epigenetic modifications can primarily promote the activity of metabolic pathways, they may also exert an inhibitory role. In this way, they participate in a complex network of interactions that regulate the metabolic behavior of malignant cells, increasing their heterogeneity and plasticity. Herein, we discuss the main epigenetic mechanisms that regulate the metabolic pathways in glioblastoma cells and highlight their targeting potential against tumor progression.

1H‑NMR‑based metabolic profiling of rat urine to assess the toxicity‑attenuating effect of the sweat‑soaking method on Radix Wikstroemia indica

Radix Wikstroemia indica (L.) C.A. Mey. (RWI) is a toxic medicinal species primarily present in the Miao area of China. The toxicity of RWI is effectively reduced whilst maintaining the therapeutic effect when processed using the ‘sweat-soaking method’, which is a common method of Traditional Chinese Medicine preparation. However, there is a lack of scientific and medical evidence to explain the potential mechanisms by which the toxicity of RWI is reduced after preparation using this method, and the endogenous systemic metabolic effect of RWI remains uncertain. The aim of the present study was to explore the endogetnous metabolic alterations caused by RWI and to examine the possibility of reducing the toxicity of RWI using the sweat-soaking method using proton nuclear magnetic resonance (NMR) metabolomic analysis in rats. Principal Component Analysis, Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA were used to assess individual proton NMR spectra. A total of 34 metabolic products were altered after delivering raw RWI, and 32 endogenous metabolites were induced by processed RWI. The metabolic pathways that lead to a significant impact on energy and carbohydrate, amino acid, organic acids and lipid metabolism following raw and processed RWI use were identified. The mitochondria of hepatic and renal tubules of rats were injured in the raw RWI group, whereas the processed product reduced or interfered with energy substrate, carbohydrate and amino acid metabolism, whilst reducing the levels of metabolic markers of hepatotoxicity and nephrotoxicity, without causing damage to the mitochondria. Our previous study showed that the median lethal dose (LD50) value of raw RWI was 4.05 g/kg in rats after oral administration; however, the LD50 value of the processed RWI could not be measured. The maximum tolerated dose and minimum lethal dose were 20 and 30 g/kg for the processed RWI, respectively, corresponding to 109 and 164 times the clinical daily dose (0.029 g/kg). Thus, the sweat-soaking method reduced the toxicity of RWI. Moreover, after processing, the toxic component YH-10 was converted into a YH-10 + OH compound, reducing the content of the toxic YH-10 by 48%, whilst also reducing the contents of the toxic components YH-12 and YH-15 by 44 and 65%, respectively. In conclusion, the present study showed that the sweat-soaking method reduced the toxicity of RWI, as evidenced by the reduction of the levels of metabolic markers and the activity of metabolic pathways, thus providing a basis for processing of RWI for clinical use.

Chloroplast pH Homeostasis for the Regulation of Photosynthesis

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.

De Novo Transcriptome of the Flagellate Isochrysis galbana Identifies Genes Involved in the Metabolism of Antiproliferative Metabolites.

Simple SummaryMicroalgae are a huge renewable and eco-friendly source of natural compounds, including vitamins, carbohydrates, pigments, sterols and lipids. They have found applications in different industrial sectors, including the pharmaceutical, nutraceutical and cosmeceutical fields. Isochrysis galbana, a marine flagellate belonging to the Haptophyta phylum, thanks to its nutraceutical composition has been proposed as a shellfish diet (Shellfish Diet 1800®) as well as in the composition of cookies, fresh pasta and yogurt. I. galbana powder, extracts or pure molecules have shown interesting bioactivities, such as antioxidant, antidiabetes and antituberculosis, while the antiproliferative activity is mainly related to lung lymphoblasts. In the current study, we aimed to identify metabolic pathways activated in stressful conditions (stationary growth phase) by transcriptomic and bioactivity-guided fractionation. Overall, the results showed antiproliferative activities against melanoma cells, suggesting new possible applications in cancer prevention and treatment.Haptophytes are important primary producers in the oceans, and among the phylum Haptophyta, the flagellate Isochrysis galbana has been found to be rich in high-value compounds, such as lipids, carotenoids and highly branched polysaccharides. In the present work, I. galbana was cultured and collected at both stationary and exponential growth phases. A transcriptomic approach was used to analyze the possible activation of metabolic pathways responsible for bioactive compound synthesis at the gene level. Differential expression analysis of samples collected at the exponential versus stationary growth phase allowed the identification of genes involved in the glycerophospholipid metabolic process, the sterol biosynthetic process, ADP-ribose diphosphatase activity and others. I. galbana raw extracts and fractions were tested on specific human cancer cells for possible antiproliferative activity. The most active fractions, without affecting normal cells, were fractions enriched in nucleosides (fraction B) and triglycerides (fraction E) for algae collected in the exponential growth phase and fraction E for stationary phase samples. Overall, transcriptomic and bioactivity data confirmed the activation of metabolic pathways involved in the synthesis of bioactive compounds giving new insights on possible Isochrysis applications in the anticancer sector.

Genetic and Drug Inhibition of LDH-A: Effects on Murine Gliomas.

Simple SummaryThree different murine glioma cell lines were modified to downregulate expression of the murine LDH-A gene using shRNA knockdown (KD) and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar in each of the cell lines. However, an unexpected increase in the aggressiveness was observed in LDH-A KD and GNE-R-140 treated GL261 intracranial gliomas, but not in CT2A and ALTS1C1 i.c. gliomas. Our results show that LDH-A KD and GNE-R-140 treated GL261 cells are better able to metabolize lactate as a primary carbon source through the TCA cycle, and are a net consumer of lactate. These results suggest that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, and that metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis may lead to the unexpected development and activation of alternative metabolic pathways resulting in enhanced tumor-cell survival in a nutrient-limited environment, leading to increased tumor aggressiveness.The effects of the LDH-A depletion via shRNA knockdown on three murine glioma cell lines and corresponding intracranial (i.c.) tumors were studied and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of genetic-shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar. LDH-A KD and GNE-R-140 unexpectedly increased the aggressiveness of GL261 intracranial gliomas, but not CT2A and ALTS1C1 i.c. gliomas. Furthermore, the bioenergetic profiles (ECAR and OCR) of GL261 NC and LDH-A KD cells under different nutrient limitations showed that (a) exogenous pyruvate is not a major carbon source for metabolism through the TCA cycle of native GL261 cells; and (b) the unique upregulation of LDH-B that occurs in GL261 LDH-A KD cells results in these cells being better able to: (i) metabolize lactate as a primary carbon source through the TCA cycle, (ii) be a net consumer of lactate, and (iii) showed a significant increase in the proliferation rate following the addition of 10 mM lactate to the glucose-free media (only seen in GL261 KD cells). Our study suggests that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, since the level of LDH-A expression and its interplay with LDH-B can lead to complex metabolic interactions between tumor cells and their environment. Metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis (e.g., inhibition of LDH-A) may lead to the unexpected development and activation of alternative metabolic pathways (e.g., upregulation of lipid metabolism and fatty-acid oxidation pathways), resulting in enhanced tumor-cell survival in a nutrient-limited environment and leading to increased tumor aggressiveness.

Atovaquone: An Inhibitor of Oxidative Phosphorylation as Studied in Gynecologic Cancers.

Simple SummaryThe Warburg effect in cancer cells (high glucose update and lactate release) is an adaptation that is considered a hallmark of most neoplasms. Blocking oxidative phosphorylation is one way to combat carcinogenesis. Atovaquone is a mitochondrial complex III inhibitor. It is currently FDA-approved for the treatment of malaria, and is a well-tolerated, orally available medication. Our laboratory studied the anti-cancer properties of atovaquone in gynecologic cancers. We found that atovaquone slowed ovarian cancer growth in both cell lines and mouse models. Additional anti-cancer effects were seen, such as the reduced proliferation of cancer stem cells and spheroids implanted in mice. Atovaquone inhibited oxygen consumption and ATP production. Metabolic studies showed that atovaquone shifted glycolysis, electronic transport and the citric acid cycle. Our studies provided the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a cancer therapy for gynecologic cancers.Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a gynecologic cancer therapeutic.

Integrating Single Cell RNAseq and Computational Modeling to Infer Hepatocyte Metabolic Functionalities during Liver Regeneration

Hepatocytes make up over 80% of liver mass and carry out all essential liver functions such as protein synthesis and detoxification, among others. Recent studies have shown distinct metabolic functions of proliferating and non‐proliferating hepatocytes as a compensatory mechanism during liver regeneration. However, this mechanism has not been studied in the case of alcoholic liver disease (ALD), which is known to disrupt the regenerative capacity and alter the metabolic processes of the liver. We seek to determine if alcohol consumption alters the balance in metabolic functionality between proliferating and non‐proliferating hepatocyte populations during liver regeneration. In the present study, rats were pair‐fed either a chronic ethanol diet or a carbohydrate control diet per Lieber DeCarli calorie‐matched liquid diet protocol. We performed 70% partial hepatectomy (PHx) and liver samples were collected at the baseline and at 24 hours post PHx. Single hepatocytes were isolated by laser capture microdissection (LCM) where the subset of proliferating hepatocytes were identified by Ki67 staining. Single‐cell RNA‐sequencing (scRNA‐seq) was employed for the purpose of identifying transcriptional differences across hepatocyte subtypes (i.e. PHx, diet, proliferation). Principal component analysis (PCA) of all samples showed distinct separation of cells into clusters based on liver regeneration (baseline and PHx samples), but not on diet (control and ethanol) and cellular proliferation (Ki67‐positive vs. negative staining). PCA of baseline samples showed separation of proliferating and non‐proliferating hepatocytes in the ethanol group. We characterized metabolic differences between hepatocyte subtypes using a computational model of genome‐scale metabolic reaction activities. We found that the metabolic compartmentalization separating proliferating from non‐proliferating hepatocytes in the carbohydrate group is absent in the ethanol group. Several functional differentiating factors arose favoring reactive oxygen species detoxification and steroid metabolism in proliferating hepatocytes from ethanol‐adapted animals at baseline, which were absent post PHx. Furthermore, proliferating cells from the carbohydrate group showed higher reaction activity in CoA synthesis, NAD metabolism and inositol phosphate metabolic pathways as compared to their non‐proliferating counterparts, both at baseline and following PHx. Our results also indicate that these pathways are also active in the non‐proliferating hepatocytes following PHx in the ethanol group. Taken together, our combinatorial transcriptomic analysis and computational modeling showed distinct metabolic pathways differentiating proliferating from non‐proliferating hepatocytes at baseline, which is absent in hepatocytes following PHx in the ethanol group. These results point towards an alternative mechanism of metabolic compensation in ethanol‐fed animals such that similar metabolic pathways are active in both proliferating and non‐proliferating hepatocytes. This lack of metabolic compartmentalization may hinder the regenerative response to PHx in the ethanol‐fed animals. Targeting the metabolic division of labor may be a potential intervention to promote liver regenerative capacity in alcoholic liver disease.

Comparative Analysis of the Metabolome and Transcriptome between the Green and Yellow-Green Regions of Variegated Leaves in a Mutant Variety of the Tree Species Pteroceltis tatarinowii.

In nature, many different factors cause plants to develop variegated leaves. To explore the mechanism of variegated leaf formation in Pteroceltis tatarinowii, a mutant variety (‘Jinyuyuan’), which was induced by ethylmethylsulfone, was selected, and its morphological structure, physiology, biochemistry, transcription and metabolism were analysed. According to differences in colour values, the colours were divided into two regions: a green region and a yellow-green region. The chlorophyll content of the two regions was significantly different. Moreover, the yellow-green regions of the leaves were significantly thinner than the green regions. The chloroplast ultrastructure in the yellow-green region revealed small chloroplasts, large vacuoles, small starch grains, obviously increased numbers of osmophilic grains, loose lamellae of the inner capsule and thin lamellae. Moreover, the yellow-green region was accompanied by oxidative stress, and the activity of the oxidative phosphorylation pathway related to oxidative activity in the transcriptome showed an upward trend. Vitamin B6 and proline contents also increased, indicating that the antioxidant activity of cells in the yellow-green region increased. Transcriptomic and metabolomic analysis showed that the differentially expressed genes (DEGs) related to chlorophyll synthesis and metabolism led to a decrease in the photosynthesis and then a decrease in the assimilation ability and contents of sucrose, starch and other assimilates. Amino acid synthesis and metabolism, lipid synthesis and the activity of metabolic pathways were obviously downregulated, and the contents of differentially accumulated metabolites associated with amino acids and lipids were also reduced. At the same time, 31 out of 32 DEGs involved in the flavonoid synthesis pathway were downregulated, which affected leaf colour. We hypothesized that the variegated leaves of P. tatarinowii ‘Jinyuyuan’ are caused by transcriptional and post-transcriptional regulation. Mutations in pigment and flavonoid synthesis pathway genes and transcription factor genes directly affect both pigment and flavonoid synthesis and degradation rate, which in turn affect carbon assimilation, carbon fixation, related protein synthesis and enzyme activity, lipid synthesis and degradation and the activity of other metabolic pathways, eventually leading to the formation of different colour regions.

A Novel Risk Score Model Based on Eleven Extracellular Matrix-Related Genes for Predicting Overall Survival of Glioma Patients.

Gliomas are the most common lethal primary brain tumors with variable survival outcomes for patients. The extracellular matrix (ECM) is linked with clinical prognosis of glioma patients, but it is not commonly used as a clinical indicator. Herein, we investigated changes in ECM-related genes (ECMRGs) via analyzing the transcriptional data of 938 gliomas from TCGA and CGGA datasets. Based on least absolute shrinkage and selection operator (LASSO) Cox regression analysis, a 11-ECMRG signature that is strongly linked with overall survival (OS) in glioma patients was identified. This signature was characterized by high-risk and low-risk score patterns. We found that the patients in the high-risk group are significantly linked with malignant molecular features and worse outcomes. Univariate and multivariate Cox regression analyses suggested that the signature is an independent indicator for glioma prognosis. The prediction accuracy of the signature was verified through time-dependent receiver operating characteristic (ROC) curves and calibration plots. Further bioinformatics analyses implied that the ECMRG signature is strongly associated with the activation of multiple oncogenic and metabolic pathways and immunosuppressive tumor microenvironment in gliomas. In addition, we confirmed that the high-risk score is an indicator for a therapy-resistant phenotype. In addition to bioinformatics analyses, we functionally verified the oncogenic role of bone morphogenetic protein 1 (BMP1) in gliomas in vitro.

Exploring the Applicability of Calorespirometry to Assess Seed Metabolic Stability Upon Temperature Stress Conditions-Pisum sativum L. Used as a Case Study.

The availability of phenotyping tools to assist breeding programs in the selection of high-quality crop seeds is of obvious interest with consequences for both seed producers and consumers. Seed germination involves the activation of several metabolic pathways, such as cellular respiration to provide the required ATP and reducing power. This work tested the applicability of calorespirometry, the simultaneous measurement of heat and CO2 rates, as a phenotyping tool to assess seed respiratory properties as a function of temperature. The effect of temperature on seed germination was evaluated after 16 h of seed imbibition by calorespirometric experiments performed in isothermal mode at 15, 20, 25, and 28°C on the seeds of three cultivars of peas (Pisum sativum L.) commonly used in conventional agriculture (cvs. ‘Rondo’, ‘Torta de Quebrar’, and ‘Maravilha d’América’). Significant differences in metabolic heat rate and CO2 production rate (RCO2) as well as in the temperature responses of these parameters were found among the three cultivars. A seed germination trial was conducted during the 6 days of imbibition to evaluate the predictive power of the parameters derived from the calorespirometric measurements. The germination trial showed that the optimal germination temperature was 20°C and low germination rates were observed at extreme temperatures (15 or 28°C). The cv. ‘Torta de Quebrar’ showed significantly higher germination in comparison with the other two cultivars at all three temperatures. In comparison with the other two cultivars, ‘Torta de Quebrar’ has the lowest metabolic heat and CO2 rates and the smallest temperature dependence of these measured parameters. Additionally, ‘Torta de Quebrar’ has the lowest values of growth rate and carbon use efficiency calculated from the measured variables. These data suggest that calorespirometry is a useful tool for phenotyping physiologic efficiency at different temperatures during early germination stages, and can determine the seeds with the highest resilience to temperature variation, in this case ‘Torta de Quebrar’.

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.

Metabolic Profiling of Thymic Epithelial Tumors Hints to a Strong Warburg Effect, Glutaminolysis and Precarious Redox Homeostasis as Potential Therapeutic Targets.

Simple SummaryThymomas and thymic carcinomas (TCs) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. This is the first metabolomics investigation on thymic epithelial tumors employing nuclear magnetic resonance spectroscopy of tissue samples. We could detect and quantify up to 37 metabolites in the major tumor subtypes, including acetylcholine that was not previously detected in other non-endocrine cancers. A metabolite-based cluster analysis distinguished three clinically relevant tumor subgroups, namely indolent and aggressive thymomas, as well as TCs. A metabolite-based metabolic pathway analysis also gave hints to activated metabolic pathways shared between aggressive thymomas and TCs. This finding was largely backed by enrichment of these pathways at the transcriptomic level in a large, publicly available, independent TET dataset. Due to the differential expression of metabolites in thymic epithelial tumors versus normal thymus, pathways related to proline, cysteine, glutathione, lactate and glutamine appear as promising therapeutic targets. From these findings, inhibitors of glutaminolysis and of the downstream TCA cycle are anticipated to be rational therapeutic strategies. If our results can be confirmed in future, sufficiently powered studies, metabolic signatures may contribute to the identification of new therapeutic options for aggressive thymomas and TCs. Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public “The Cancer Genome Atlas” (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other non-endocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong “Warburg effect”, glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.

Effect of various factors on ejaculate fertility in vitro

Introduction. Current demographic situation in Russia is characterized by decreasing birth rate. According to the World Health Organization, percentage of child-free marriages in various countries is 10–15 %. In Russia, the National Medical Research Center for Obstetrics, Gynecology, and Perinatology named after Academician V.I. Kulakov states that this number is 17 %. More than 4 million men suffer from infertility of various types. In recent years, pathologies of male reproductive function have achieved medical and social significance due to progressively decreasing sperm fertility. Fertility disorders are considered multi-factor conditions caused by internal and external factors and leading to pathological changes in sex organs. Correction of pathologies of male fertility does not always lead to positive results. Therefore, it is important to develop and study effective pharmaceuticals affecting the main fertility parameters.The study objective is to investigate the effect of various biological pharmaceuticals, physical and chemical factors on parameters of ejaculate fertility in vitro.Materials and methods. Experiments with a protein-peptide complex (PPC), methylene blue, and hydrogen peroxide were performed on human semen. After semen dilution, the sample was studied under the microscope and sperm motility and other ejaculate parameters were evaluated per the 5th edition WHO standard. Experiments were performed at 20–22 ºС. Statistical data analysis was performed using the Student’s t-test. Differences were considered statistically significant at р <0.05.Results. The results show increased motility in the fraction of active motile sperm in first 30 minutes after incubation with methylene blue. In case of initial asthenospermia, active motility increased by 72 %, in case of normospermia by 89 %. After 2 hours, all motility fractions were at the baseline level.The experiments also showed significant changes in sperm motility in the presence of PPC preparation in the ejaculate: increase in sperm motility was observed beginning at 30-minute mark and this level persisted through 3 hours of observation. A more pronounced change, by 60 %, was observed in the active motile sperm; total motility increased by up to 30 %. After 24 hours, sperm motility remained close to the baseline level, the number of normal sperm forms and live cells did not change. Dependence of the motility change on the preparation concentration should be noted: the highest increase was observed at PPC concentration with total protein level of 10–12 mg/mL. Higher concentrations did not have a positive effect on sperm motility. In experiments with low hydrogen peroxide concentration, a positive effect on sperm motility was observed.Conclusions. Sperm motility is supported by glycolysis energy, and one of the glycolysis enzymes glyceraldehyde 3-phosphate dehydrogenase is tightly bound to the fibrous layer of the flagellum, activation of metabolic pathways leading to increased enzyme activity, increased sperm motility. The mechanism of the observed effect is not entirely clear, however, the obtained data demonstrate potential benefits of further studies on use of pharmaceuticals in andrological and reproductive practice, assisted reproductive technologies, as well as for stimulation of sperm motility in further experimental studies.

Metabolomic Pathway Activity with Genomic Single-Nucleotide Polymorphisms Associated with Colorectal Cancer Recurrence and 5-Year Overall Survival.

Metabolomic analysis in colorectal cancer (CRC) is an emerging research area with both prognostic and therapeutic targeting potential. We aimed to identify metabolomic pathway activity prognostic for CRC recurrence and overall survival and cross-reference such metabolomic data with prognostic genomic single-nucleotide polymorphisms (SNPs). A systematic search of PubMed, Embase and Cochrane Library was performed for studies reporting prognostic metabolomic pathway activity in CRC in keeping with PRISMA guidelines. The QUADOMICS tool was used to assess study quality. MetaboAnalyst software (version4.0) was used to map metabolites that were associated with recurrence and survival in CRC to recognise metabolic pathways and identify genomic SNPs associated with CRC prognosis, referencing the following databases: Human Metabolome Database (HMDB), the Small Molecule Pathway Database (SMPDB), PubChem and Kyoto Encyclopaedia of Genes and Genomes (KEGG) Pathway Database. Nine studies met the inclusion criteria, reporting on 1117 patients. Increased metabolic activity in the urea cycle (p = 0.002, FDR = 0.198), ammonia recycling (p = 0.004, FDR = 0.359) and glycine and serine metabolism (p = 0.004, FDR = 0.374) was prognostic of CRC recurrence. Increased activity in aspartate metabolism (p < 0.001, FDR = 0.079) and ammonia recycling (p = 0.004, FDR = 0.345) was prognostic of survival. Eight resulting SNPs were prognostic for CRC recurrence (rs2194980, rs1392880, rs2567397, rs715, rs169712, rs2300701, rs313408, rs7018169) and three for survival (rs2194980, rs169712, rs12106698) of which two overlapped with recurrence (rs2194980, rs169712). With a caveat on study heterogeneity, specific metabolites and metabolic pathway activity appear evident in the setting of poor prognostic colorectal cancers and such metabolic signatures are associated with specific genomic SNPs.

Pharmacology and toxicology of tannins.

Tannins are an interesting class of polyphenols, characterized, in almost all cases, by a different degree of polymerization, which, inevitably, markedly influences their bioavailability, as well as biochemical and pharmacological activities. They have been used for the process of tanning to transform hides into leather, from which their name derives. For several time, they have not been accurately evaluated, but now researchers have started to unravel their potential, highlighting anti-inflammatory, antimicrobial, antioxidant and anticancer activities, as well as their involvement in cardiovascular, neuroprotective and in general metabolic diseases prevention. The mechanisms underlying their activity are often complex, but the main targets of their action (such as key enzymes modulation, activation of metabolic pathways and changes in the metabolic fluxes) are highlighted in this review, without losing sight of their toxicity. This aspect still needs further and better-designed study to be thoroughly understood and allow a more conscious use of tannins for human health.

A248 THE ROLE OF DIETARY TRYPTOPHAN IN INDOLE AND KYNURENINE PRODUCTION AND IMMUNE MODULATION IN HEALTHY INDIVIDUALS

BackgroundNatural supplements are widely consumed by the general public, with little evidence of mechanistic support. Tryptophan has gained central attention, being transformed by host and gut microbial enzymes into multiple bioactive metabolites that regulate immunity and mood. Indoles are activators of the aryl hydrocarbon receptor (AhR), crucial for the maintenance of intestinal homeostasis. Tryptophan has been advocated to prevent chronic inflammatory conditions, however the clinical data to support this are missing.AimsTo investigate clinical, immune, and metabolic parameters in response to tryptophan supplementation, in healthy subjects on a low tryptophan diet.MethodsWe performed a randomized, double blind, placebo-controlled crossover study in 20 healthy volunteers (18 - 75 years old). Subjects were instructed to start a low tryptophan diet and then randomly assigned to a 3-week tryptophan supplementation (3g/day) or placebo, in enteric coated capsules. After a 2-week washout period, subjects crossed over to the opposite intervention arm. Questionnaires were used to assess bowel symptoms, anxiety, depression and stress levels (GSRS, HADS and DASS21, respectively). Stool, urine, blood and duodenal aspirates were collected to measure tryptophan metabolites and cytokines.ResultsTryptophan supplementation had no changes in gastrointestinal symptoms or behavioral parameters. Compared with placebo, tryptophan increased urinary and plasma levels of indoleamine 2,3-dioxygenase/kynurenine (p= 0.002 and p= 0.02, respectively) and indoles (p= 0.001 and p= 0.01, respectively), suggestive of activation of host and microbial metabolic pathways. Urinary and plasma metabolites were higher than in feces (p=<0.05), suggesting their active absorption in the small intestine. There were no differences in AhR activity in duodenal aspirates or in stool. Although no changes in the cytokine production were detected, serum kynurenine pathway metabolites negatively correlated with IL-8 levels (R=-0.72; p=0.001). Fecal tryptophan metabolites levels positively correlated with anxiety and depression scores, suggesting that the microbial metabolism of dietary tryptophan in the colon impacts host behavior.ConclusionsTryptophan supplementation in healthy individuals was safe and had a measurable influence on microbial and host metabolism, mainly kynurenine and indole pathways, with known immunomodulatory properties. Tryptophan was metabolized and absorbed in the small intestine, reflected by the high metabolite levels in plasma and urine. Fecal metabolites correlating with clinical parameters reflect subjects’ long-term diet. Further studies are warranted to study tryptophan supplementation in disorders with altered AhR pathways.Funding AgenciesCIHR

Lipidomic profiling in patients with heavily pretreated castration-resistant prostate cancer.

174 Background: Despite the advent of chemotherapy and androgen-receptor signaling inhibitors (ARSi), patients with metastatic castration-resistant prostate cancer (mCRPC) still show poor prognosis and reduced survival. The selective pressure induced by treatments favors the activation of alternative metabolic pathways that allow the persistence of cancer cells in unfavorable conditions. This study aimed at assessing the lipidomic profiles of patients with mCRPC, in order to identify lipid species potentially useful to predict for prognosis and response to therapies. Methods: Plasma samples were collected from patients with mCRPC who were starting a first-line treatment for mCRPC (1L) (n = 30) and from those who had already received at least two lines of treatment for mCRPC ( &gt; 2L) (n = 19), including at least one ARSi and a taxane. Lipids were extracted from plasma samples using a modified Matyash method employing a mix of cold MeOH and MTBE and containing a mix of deuterated standards (Splash Lipidomix). Lipids were then analyzed with an untargeted lipidomic approach using an UHPLC Vanquish system coupled with a Q-Exactive Plus instrument. T-test was then applied to identify lipid species and classes that were differentially expressed in 1L vs &lt; 2L patients. Results: We identified and quantified a total of 907 plasma lipids. Overall, 68 lipid species were significantly dysregulated in &gt; 2L compared to 1L plasma samples. 56 species were found to be upregulated, whereas 12 were downregulated, with a fold change (FC) &gt; 1.3 or &lt; 0.75 and a p-value &lt; 0.05. At the level of lipid classes, &gt; 2L patients showed higher levels of acylcarnitine (CAR), diacylglycerols (DG), phosphatidylethanolamine (PE) and triacylglycerols (TG). The following lipids showed the highest FC: DG28:2 = 4.2; CAR14:0 = 3.7; CAR20:1 = 2.6; CAR18:0 = 2.3; PE40:6 = 2.3. Conversely, significantly lower levels of specific phosphatidylcholines (PC) and sterols (ST) were found in &gt; 2L compared to 1L patients. The following species showed the lowest FC: PCO-39:3 = 0.52; ST29:1;O;S = 0.55; PC36:5 = 0.55; PC36.4 = 0.60. These results suggest that patients with strongly pretreated mCRPC show higher levels of lipid species involved in the switch between the glucose and fatty acid metabolism. We also found a dysregulation of ceramides and sphingomyelins which are well-known lipid species involved in apoptosis and cancer progression. Specific lipids warrant further investigation to be used as potential prognostic and predictive biomarkers in patients with mCRPC. Conclusions: Using a quantitative mass spectrometry approach, we investigated the dysregulation of lipids and lipid metabolism in mCRPC patients at different disease stages. We found that specific lipid species show differential levels in patients pretreated with ARSi and chemotherapy, compared to those who are naïve to these treatments, paving the way for further investigations in this field.

Seasonal variations in the composition and functional profiles of gut microbiota reflect dietary changes in plateau pikas.

Seasonal variations in gut microbiota of small mammals and how they are influenced by environmental variables are relatively poorly understood. We sampled 162 wild plateau pikas (Ochotona curzoniae) in 4 seasons over 2 and a half years and recorded the air temperature, precipitation, and nutrient content in edible vegetation at the sampling site. After conducting 16S rRNA and shotgun metagenomic sequencing, we found that the highest alpha diversity, the relative abundance of Firmicutes, and the simplest co‐occurrence network occurred in winter, whereas the highest relative abundance of Proteobacteria and the most complex network structure were observed in spring. The highest relative abundance of Verrucomicrobiota and Spirochaetota was seen in summer and autumn, respectively. Air temperature, precipitation, and the contents of crude protein, crude fiber, and polysaccharide in vegetation had significant effects on the seasonal changes in gut microbiota. Diet contributed more to microbial variation than climatic factors. Metagenomic analysis revealed that the amino acid metabolism pathway and axillary activity enzymes were most abundant in summer, while abundance of carbohydrate‐binding modules and carbohydrate esterases was highest in spring. These microbial variations were related to the changes in dietary nutrition, indicating that gut microbiota of plateau pika contribute to the efficient use of food resources. This study provides new evidence of how external environmental factors affect the intestinal environment of small mammals.

Abstract P5-05-05: Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells

Abstract Introduction. With the goal of defining the in-breast environment that facilitates the development of estrogen receptor negative (ER-) breast cancer (BC), we have previously identified a set of genes involved in lipid metabolism with increased expression in the contralateral unaffected breast of women with ER- BC (PMID: 28263391). MCF-10A cells exposed to lipids in vitro display profound changes in gene expression, chromatin packing density, chromatin accessibility and histone posttranslational modifications (PTMs). In addition, flux through thirty-eight metabolic reactions was significantly increased by this exposure, including those of the electron transport chain (ETC). However, the mechanisms by which lipids induce molecular changes that result in epigenomic reprogramming, and potentially promote malignant transformation remain to be elucidated. We hypothesized that dysfunction of the ETC and increased serine, glycine, one carbon (SGOC) metabolism pathway activity play a role in the modulation of epigenetics. Methods. To interrogate potential mechanisms that link lipids to ER- BC genesis, estrogen and progesterone receptor negative MCF-10A cells were cultured in MEBM/MEGM and exposed to vehicle, octanoic acid (C8) or linoleic acid (C18) for 24 hours. Histone PTMs were assayed by liquid chromatography-mass spectrometry. Gene expression was measured by RNA-seq and differentially expressed genes between vehicle and lipid treated groups were identified using the DESeq2 R package. ATAC-seq was performed to study chromatin accessibility. MCF-10A cells were exposed to C8 in presence or absence of inhibitors of Complex I (Metformin) and Complex III (Antimycin A) of the ETC. Real-time qPCR was performed to measure epigenetic modulators, Unfolded Protein Response (UPR) pathway and SGOC metabolism genes. Results. Histone PTMs changed significantly following 24 hours exposure to C8 or C18. Exposure to C8 resulted in the modulation of the expression of genes involved in the UPR/recognition of mitochondrial dysfunction (ATF4), de novo serine biosynthesis (PHGDH, PSAT1, SPH) and one-carbon metabolism (SHMT2, MTHFD2) pathways as well as numerous genes coding for epigenetic modulators. Upon C18 exposure, the binding motif for ATF4 was among the most significantly enriched motifs in the open chromatin regions and p53 was among the motifs with significantly decreased accessibility. Interestingly ATF4 actives PHGDH expression while p53 suppresses it. Exposure to C8 in presence of ETC inhibitors affected gene expression in the same direction as lipid exposure and co-treatment had an additive effect. Fatty acid oxidation and inhibition of the ETC increased the expression of PHGDH: C8, 2.6-fold (p-value = 0.005); Metformin, 2.3-fold (p-value = 0.0052); Antimycin A, 5.1-fold (p-value &amp;lt; 0.0001); C8 plus Metformin, 5.3-fold (p-value &amp;lt; 0.0001), and C8 plus Antimycin A, 5.2-fold (p-value &amp;lt; 0.0001). Conclusions. The modulation of epigenetic effectors does not appear to be a consequence of increased flux through the ETC but rather a consequence of increased SGOC metabolism, and the methionine cycle. Our in vitro model shows that lipid-induced stress increases ATF4 expression. ATF4 controls the expression of PHGDH, PSAT1, SPH, SHMT2 and MTHFD2 which influences epigenetics through S-adenosyl methionine production, the major methyl donor. PHGDH overexpression appears to be the key in our model as it has been associated with ER- BC and its engineered overexpression in non-transformed breast cells causes alterations associated with malignant transformation. The increase in PDGDH expression is hypothesized to compensate for mitochondrial dysfunction and to restore redox homeostasis by producing precursors that can be used for the production of antioxidants, such as glutathione. Citation Format: Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-05.