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Givosiran is a subcutaneously administered, liver-targeted RNA interference (RNAi) therapeutic that has been approved for treating acute hepatic porphyria (AHP). Elevation in plasma homocysteine (hyperhomocysteinemia) has been reported in AHP patients, and treatment with givosiran has been reported to further increase homocysteine levels in some patients. The mechanism of homocysteine elevation during givosiran treatment is unknown, but has been hypothesized to be mediated by a reduction in activity of cystathionine ß-synthase (CBS), which uses homocysteine as a substrate. A liquid chromatography-tandem mass spectrometry-based assay was adapted to measure circulating CBS activity. Using plasma collected from the Phase III ENVISION study, CBS activity was measured to directly evaluate whether it is associated with elevated homocysteine levels in givosiran-treated patients. CBS activity was reduced following givosiran treatment and both homocysteine and methionine levels were inversely correlated with CBS activity. Following administration of a supplement containing vitamin B6, a cofactor for CBS, in four patients during the trial, plasma CBS activity was found to increase, mirroring a corresponding decrease in homocysteine levels. These results support the hypothesis that elevated homocysteine levels following givosiran treatment result from a reduction of CBS activity and that vitamin B6 supplementation lowers homocysteine levels by increasing CBS activity.
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Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.
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Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.
Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Fator de Crescimento Epidérmico/genética , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Mama/crescimento & desenvolvimento , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/patologia , Células Tumorais CultivadasRESUMO
Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.
Assuntos
Aldeído Oxirredutases/fisiologia , Carbolinas/farmacologia , Cicloexilaminas/farmacologia , Ferroptose/efeitos dos fármacos , Hematopoese/fisiologia , Leucemia Mieloide Aguda/enzimologia , Proteínas de Neoplasias/fisiologia , Fenilenodiaminas/farmacologia , Aldeído Oxirredutases/genética , Aldeídos/farmacologia , Animais , Linhagem Celular Tumoral , Citarabina/administração & dosagem , Doxorrubicina/administração & dosagem , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Ácido Oleico/farmacologia , Proteínas de Fusão Oncogênica/fisiologia , Oxirredução , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/fisiologiaRESUMO
Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.
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Metabolismo Energético , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glicólise , Fosfatidilinositol 3-Quinases/análise , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Quinases Ativadas por AMP/análise , Linhagem Celular , Proliferação de Células , Humanos , NAD/análiseRESUMO
D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.
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Ciclina D3/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , Glicólise/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fosfofrutoquinase-1/metabolismo , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Purinas/farmacologia , Purinas/uso terapêutico , Piruvato Quinase/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer metabolism has emerged as an indispensable part of contemporary cancer research. During the past 10 years, the use of stable isotopic tracers and network analysis have unveiled a number of metabolic pathways activated in cancer cells. Here, we review such pathways along with the particular tracers and labeling observations that led to the discovery of their rewiring in cancer cells. The list of such pathways comprises the reductive metabolism of glutamine, altered glycolysis, serine and glycine metabolism, mutant isocitrate dehydrogenase (IDH) induced reprogramming and the onset of acetate metabolism. Additionally, we demonstrate the critical role of isotopic labeling and network analysis in identifying these pathways. The alterations described in this review do not constitute a complete list, and future research using these powerful tools is likely to discover other cancer-related pathways and new metabolic targets for cancer therapy.
Assuntos
Aminoácidos/metabolismo , Glicólise , Isocitrato Desidrogenase/metabolismo , Marcação por Isótopo/métodos , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Aminoácidos/genética , Animais , Humanos , Isocitrato Desidrogenase/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologiaRESUMO
Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo.
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Aminoácidos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Pinocitose , Proteólise , Albumina Sérica/metabolismo , Albuminas/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia Gasosa , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Isótopos de Nitrogênio , Plasmaferese , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Carcinoma de Célula de Merkel/virologia , Transformação Celular Viral/fisiologia , Glicólise/genética , Neoplasias Cutâneas/virologia , Carcinoma de Célula de Merkel/metabolismo , Linhagem Celular , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Poliomavírus das Células de Merkel , Metabolômica , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/metabolismo , TransfecçãoRESUMO
BACKGROUND: The study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism. RESULTS: Using a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD(+), acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux. CONCLUSIONS: Our analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD(+), while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.
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Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Microambiente Tumoral , Animais , Glicemia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Glucose/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Transplante de Neoplasias , Proteínas Proto-Oncogênicas p21(ras)/genética , Ácido Pirúvico/metabolismoRESUMO
Glutamine has recently emerged as a key substrate to support cancer cell proliferation, and the quantification of its metabolic flux is essential to understand the mechanisms by which this amino acid participates in the metabolic rewiring that sustains the survival and growth of neoplastic cells. Glutamine metabolism involves two major routes, glutaminolysis and reductive carboxylation, both of which begin with the deamination of glutamine to glutamate and the conversion of glutamate into α-ketoglutarate. In glutaminolysis, α-ketoglutarate is oxidized via the tricarboxylic acid cycle and decarboxylated to pyruvate. In reductive carboxylation, α-ketoglutarate is reductively converted into isocitrate, which is isomerized to citrate to supply acetyl-CoA for de novo lipogenesis. Here, we describe methods to quantify the metabolic flux of glutamine through these two routes, as well as the contribution of glutamine to lipid synthesis. Examples of how these methods can be applied to study metabolic pathways of oncological relevance are provided.
Assuntos
Bioquímica/métodos , Ácido Glutâmico/análise , Glutamina/análise , Glutamina/metabolismo , Neoplasias/metabolismo , Isótopos de Carbono , Hipóxia Celular , Células Cultivadas , Cromatografia Gasosa-Espectrometria de Massas , Ácido Glutâmico/metabolismo , Humanos , Lipídeos/biossínteseRESUMO
Reductively metabolized glutamine is a major cellular carbon source for fatty acid synthesis during hypoxia or when mitochondrial respiration is impaired. Yet, a mechanistic understanding of what determines reductive metabolism is missing. Here we identify several cellular conditions where the α-ketoglutarate/citrate ratio is changed due to an altered acetyl-CoA to citrate conversion, and demonstrate that reductive glutamine metabolism is initiated in response to perturbations that result in an increase in the α-ketoglutarate/citrate ratio. Thus, targeting reductive glutamine conversion for a therapeutic benefit might require distinct modulations of metabolite concentrations rather than targeting the upstream signalling, which only indirectly affects the process.
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Células/metabolismo , Ácido Cítrico/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Acetatos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Ácidos Graxos/metabolismo , Humanos , Ácido Láctico/metabolismo , Modelos Biológicos , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Oxirredução , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer.
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Glucose/metabolismo , Glutamina/metabolismo , Metformina/farmacologia , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Oxirredução , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologiaRESUMO
The metabolic engineer's toolbox, comprising stable isotope tracers, flux estimation and analysis, pathway identification, and pathway kinetics and regulation, among other techniques, has long been used to elucidate and quantify pathways primarily in the context of engineering microbes for producing small molecules of interest. Recently, these tools are increasingly finding use in cancer biology due to their unparalleled capacity for quantifying intracellular metabolism of mammalian cells. Here, we review basic concepts that are used to derive useful insights about the metabolism of tumor cells, along with a number of illustrative examples highlighting the fundamental contributions of these methods to elucidating cancer cell metabolism. This area presents unique opportunities for metabolic engineering to expand its portfolio of applications into the realm of cancer biology and help develop new cancer therapies based on a new class of metabolically derived targets.