ABSTRACT
Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows great but incomplete benefits, underscoring the need to better understand the roles of glutamine and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria across diverse cell types, excluding ccRCC. GLS1 clustering is rapid (1-3 hours) and reversible, is specifically driven by the level of intracellular glutamate, and is mediated by mitochondrial fission. Clustered GLS1 has markedly enhanced glutaminase activity and promotes cell death under glutamine-deprived conditions. We further show that HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby protecting ccRCC cells from cell death induced by glutamine deprivation. Reversing this protection, by genetic expression of GLS1 mutants that constitutively cluster, enhances ccRCC cell death in culture and suppresses ccRCC growth in vivo . These finding provide multiple insights into cellular glutamine handling, including a novel metabolic pathway by which HIF2α promotes ccRCC, and reveals a potential therapeutic avenue to synergize with HIF2α inhibition in the treatment of ccRCC.
ABSTRACT
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
Subject(s)
ATP Citrate (pro-S)-Lyase , Alternative Splicing , Neoplasms , Humans , Animals , Mice , ATP Citrate (pro-S)-Lyase/metabolism , ATP Citrate (pro-S)-Lyase/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Phenotype , Exons , AcetylationABSTRACT
Branched chain ketoacid dehydrogenase kinase (BCKDK) deficiency is a recently described inherited neurometabolic disorder of branched chain amino acid (BCAA) metabolism implying increased BCAA catabolism. It has been hypothesized that a severe reduction in systemic BCAA levels underlies the disease pathophysiology, and that BCAA supplementation may ameliorate disease phenotypes. To test this hypothesis, we characterized a recent mouse model of BCKDK deficiency and evaluated the efficacy of enteral BCAA supplementation in this model. Surprisingly, BCAA supplementation exacerbated neurodevelopmental deficits and did not correct biochemical abnormalities despite increasing systemic BCAA levels. These data suggest that aberrant flux through the BCAA catabolic pathway, not just BCAA insufficiency, may contribute to disease pathology. In support of this conclusion, genetic re-regulation of BCAA catabolism, through Dbt haploinsufficiency, partially rescued biochemical and behavioral phenotypes in BCKDK deficient mice. Collectively, these data raise into question assumptions widely made about the pathophysiology of BCKDK insufficiency and suggest a novel approach to develop potential therapies for this disease.
ABSTRACT
Branched chain ketoacid dehydrogenase kinase (BCKDK) deficiency is a recently described inherited neurometabolic disorder of branched chain amino acid (BCAA) metabolism implying increased BCAA catabolism. It has been hypothesized that a severe reduction in systemic BCAA levels underlies the disease pathophysiology, and that BCAA supplementation may ameliorate disease phenotypes. To test this hypothesis, we characterized a recent mouse model of BCKDK deficiency and evaluated the efficacy of enteral BCAA supplementation in this model. Surprisingly, BCAA supplementation exacerbated neurodevelopmental deficits and did not correct biochemical abnormalities despite increasing systemic BCAA levels. These data suggest that aberrant flux through the BCAA catabolic pathway, not just BCAA insufficiency, may contribute to disease pathology. In support of this conclusion, genetic re-regulation of BCAA catabolism, through Dbt haploinsufficiency, partially rescued biochemical and behavioral phenotypes in BCKDK deficient mice. Collectively, these data raise into question assumptions widely made about the pathophysiology of BCKDK insufficiency and suggest a novel approach to develop potential therapies for this disease.
ABSTRACT
Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.
ABSTRACT
The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake into mitochondria. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca 2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase, a modest increase of basal mitochondrial respiration and a significant increase of acute Ca 2+ -dependent stimulation of mitochondrial respiration. Inhibition of mitochondrial Ca 2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo , and slower cell proliferation in vitro associated with delayed progression through S-phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca 2+ uptake for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation.
ABSTRACT
Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.
Subject(s)
Acyl Coenzyme A/metabolism , Cell Compartmentation , Cell Nucleus/metabolism , Energy Metabolism , Histones/metabolism , Metabolomics , Protein Processing, Post-Translational , Animals , Cell Differentiation , Chromatography, Liquid , Cytosol/metabolism , Epigenesis, Genetic , Hep G2 Cells , Humans , Isoleucine , Metabolome , Mice , Mitochondria/metabolism , Oxygen/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
Insulin stimulates adipose tissue to extract fatty acids from circulation and sequester them inside adipose cells. How fatty acids are transported across the capillary endothelial barrier, and how this process is regulated, remains unclear. We modeled the relationship of adipocytes and endothelial cells in vitro to test the role of insulin in fatty acid transport. Treatment of endothelial cells with insulin did not affect endothelial fatty acid uptake, but endothelial cells took up more fatty acids when exposed to medium conditioned by adipocytes treated with insulin. Manipulations of this conditioned medium indicated that the secreted factor is a small, hydrophilic, non-proteinaceous metabolite. Factor activity was correlated with lactate concentration, and inhibition of lactate production in adipocytes abolished the activity. Finally, lactate alone was sufficient to increase endothelial uptake of both free fatty acids and lipids liberated from chylomicrons, and to promote transendothelial transport, at physiologically relevant concentrations. Taken together, these data suggest that insulin drives adipocytes to secrete lactate, which then acts in a paracrine fashion to promote fatty acid uptake and transport across the neighboring endothelial barrier.
Subject(s)
Fatty Acids , Insulin , Adipocytes , Endothelial Cells , Endothelium, Vascular , Glucose , Lactic AcidABSTRACT
Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.
Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Glutamine/deficiency , Hexosamines/metabolism , Pancreatic Neoplasms/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Cell Line , Humans , Mice , Mice, NudeABSTRACT
In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.
ABSTRACT
BACKGROUND AND AIMS: Advances in cancer treatment have improved survival; however, local recurrence and metastatic disease-the principal causes of cancer mortality-have limited the ability to achieve durable remissions. Local recurrences arise from latent tumor cells that survive therapy and are often not detectable by conventional clinical imaging techniques. Local recurrence after transarterial embolization (TAE) of hepatocellular carcinoma (HCC) provides a compelling clinical correlate of this phenomenon. In response to TAE-induced ischemia, HCC cells adapt their growth program to effect a latent phenotype that precedes local recurrence. APPROACH AND RESULTS: In this study, we characterized and leveraged the metabolic reprogramming demonstrated by latent HCC cells in response to TAE-induced ischemia to enable their detection in vivo using dynamic nuclear polarization (DNP) magnetic resonance spectroscopic imaging (MRSI) of 13 carbon-labeled substrates. Under TAE-induced ischemia, latent HCC cells demonstrated reduced metabolism and developed a dependence on glycolytic flux to lactate. Despite the hypometabolic state of these cells, DNP-MRSI of 1-13 C-pyruvate and its downstream metabolites, 1-13 C-lactate and 1-13 C-alanine, predicted histological viability. CONCLUSIONS: These studies provide a paradigm for imaging latent, treatment-refractory cancer cells, suggesting that DNP-MRSI provides a technology for this application.
Subject(s)
Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/therapy , Embolization, Therapeutic , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/therapy , Magnetic Resonance Imaging/methods , Magnetic Resonance Spectroscopy , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Disease Models, Animal , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Rats , Rats, WistarABSTRACT
While patient-derived xenograft (PDX) models of hepatocellular carcinoma (HCC) have been successfully generated from resected tissues, no reliable methods have been reported for the generation of PDXs from patients who are not candidates for resection and represent the vast majority of patients with HCC. Here we compare two methods for the creation of PDXs from HCC biopsies and find that implantation of whole biopsy samples without the addition of basement membrane matrix favors the formation of PDX tumors that resemble Epstein-Barr virus (EBV)-driven B-cell lymphomas rather than HCC tumors. In contrast, implantation with Matrigel supports growth of HCC cells and leads to a high rate of HCC tumor formation from these biopsies. We validate the resulting PDXs, confirm their fidelity to the patients' disease and conclude that minimally invasive percutaneous liver biopsies can be used with relatively high efficiency to generate PDXs of HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Animals , Carcinoma, Hepatocellular/virology , Collagen , Drug Combinations , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/isolation & purification , Heterografts , Humans , Image-Guided Biopsy , Laminin , Liver Neoplasms/virology , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/virology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation/methods , Proteoglycans , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: To establish the capability of near-infrared fluorescence (NIRF) imaging for the detection of matrix metalloproteinase 2 (MMP-2) activity as a biomarker of vascular remodeling (VR) in arteriovenous fistulae (AVFs) in vivo. MATERIALS AND METHODS: AVFs were created in the right groins of Wistar rats (n = 10), and sham procedures were performed in the contralateral groins. Fistulography via a left common carotid artery approach confirmed stenosis (> 50%) in a subset of animals (n = 5) 4 weeks after AVF creation. After administration of MMP-2-activated NIRF probe, near-infrared imaging was performed in vivo and ex vivo of both the AVF and the sham-treated vessels to measure radiant efficiency of MMP-2-activated NIRF signal over background. Histologic analyses of AVF and sham-treated vessels were performed to measure VR defined as inward growth of the vessel caused by intimal thickening. RESULTS: AVFs demonstrated a significantly higher percentage increase in radiant efficiency over background compared with sham vessels (45.5 ± 56% vs 16.1 ± 17.8%; P = .008). VR in AVFs was associated with increased thickness of neointima staining positively for MMP-2 (161.8 ± 45.5 µm vs 73.2 ± 36.7 µm; P = .01). A significant correlation was observed between MMP-2 activity as measured by relative increase in radiant efficiency for AVFs and thickness of neointima staining positively for MMP-2 (P = .039). CONCLUSIONS: NIRF imaging can detect increased MMP activity in remodeled AVFs compared with contralateral sham vessels. MMP-2-activated NIRF signal correlates with the severity of intimal thickening. These findings suggest NIRF imaging of MMP-2 may be used as a biomarker of the vascular remodeling underlying stenosis.
Subject(s)
Arteriovenous Shunt, Surgical/methods , Femoral Artery/diagnostic imaging , Femoral Vein/diagnostic imaging , Graft Occlusion, Vascular/diagnostic imaging , Groin/blood supply , Matrix Metalloproteinase 2/metabolism , Optical Imaging/methods , Spectroscopy, Near-Infrared , Vascular Remodeling , Animals , Arteriovenous Shunt, Surgical/adverse effects , Biomarkers/metabolism , Femoral Artery/enzymology , Femoral Artery/physiopathology , Femoral Artery/surgery , Femoral Vein/enzymology , Femoral Vein/physiopathology , Femoral Vein/surgery , Fluorescent Dyes/administration & dosage , Graft Occlusion, Vascular/enzymology , Graft Occlusion, Vascular/physiopathology , Models, Animal , Neointima , Predictive Value of Tests , Rats, Wistar , Renal DialysisABSTRACT
BACKGROUND: The gut microbiota is now known to play an important role contributing to inflammatory-based chronic diseases. This study examined intestinal integrity/inflammation and the gut microbial communities in sedentary and exercising mice presented with a normal or high-fat diet. METHODS: Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-weeks and randomly assigned to exercise or sedentary groups. After 12 weeks animals were sacrificed and duodenum/ileum tissues were fixed for immunohistochemistry for occludin, E-cadherin, and cyclooxygenase-2 (COX-2). The bacterial communities were assayed in fecal samples using terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of 16S rRNA gene amplicons. RESULTS: Lean sedentary (LS) mice presented normal histologic villi while obese sedentary (OS) mice had similar villi height with more than twice the width of the LS animals. Both lean (LX) and obese exercise (OX) mice duodenum and ileum were histologically normal. COX-2 expression was the greatest in the OS group, followed by LS, LX and OX. The TRFLP and pyrosequencing indicated that members of the Clostridiales order were predominant in all diet groups. Specific phylotypes were observed with exercise, including Faecalibacterium prausnitzi, Clostridium spp., and Allobaculum spp. CONCLUSION: These data suggest that exercise has a strong influence on gut integrity and host microbiome which points to the necessity for more mechanistic studies of the interactions between specific bacteria in the gut and its host.