Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the ...electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.
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•Use of 2H stable isotopes to trace NAD(P)H metabolism in cells•Quantification of pentose phosphate pathway contribution to cytosolic NADPH•Reporter system to distinguish cytosolic and mitochondrial NADPH•Resolve direction of otherwise identical compartmentalized redox reactions
NAD(P)H cofactors are not transported across cell membranes, with metabolic shuttles required for transport of reducing equivalents between the mitochondria and cytosol. Lewis et al. develop an approach to resolve NADPH-dependent pathways in these distinct metabolic compartments and show that directionality of serine/glycine interconversions can be determined within intact cells.
Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in ...intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied 13C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.
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•Oxidation of fatty acids and amino acids is increased upon MPC inhibition•Respiration, proliferation, and biosynthesis are maintained when MPC is inhibited•Glutaminolytic flux supports lipogenesis in the absence of MPC•MPC inhibition is distinct from hypoxia or complex I inhibition
Vacanti et al. describe the inherent metabolic flexibility of mammalian cells. In response to genetic or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC), myocytes and cancer cells maintain respiration via oxidation of fatty acids and amino acids. These results highlight a potential mechanism for control of mitochondrial substrate utilization.
Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a ...property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.
Continuous de novo fatty acid synthesis is a common feature of cancer that is required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme ...acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain the de novo fatty acid synthesis needed for growth and viability of non-small-cell lung cancer (NSCLC) cells. We describe the ability of ND-646-an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization-to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53
(also known as KRAS p53) and Kras;Stk11
(also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology.
Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. ...Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown. Here we show that Ras-transformed cells use macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.
Specific point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) occur in a variety of cancers, including acute myeloid leukemia (AML), low-grade gliomas, and chondrosarcomas. These ...mutations inactivate wild-type enzymatic activity and convey neomorphic function to produce d-2-hydroxyglutarate (d-2HG), which accumulates at millimolar levels within tumors. d-2HG can impact α-ketoglutarate-dependent dioxygenase activity and subsequently affect various cellular functions in these cancers. Inhibitors of the neomorphic activity of mutant IDH1 and IDH2 are currently in Phase I/II clinical trials for both solid and blood tumors. As IDH1 and IDH2 represent key enzymes within the tricarboxylic acid (TCA) cycle, mutations have significant impact on intermediary metabolism. The loss of some wild-type metabolic activity is an important, potentially deleterious and therapeutically exploitable consequence of oncogenic IDH mutations and requires continued investigation in the future. Here we review how IDH1 and IDH2 mutations influence cellular metabolism, epigenetics, and other biochemical functions, discussing these changes in the context of current efforts to therapeutically target cancers bearing these mutations.
Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here, we demonstrate that both ...glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth, chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration, altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development.
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•Medium choice influences the metabolic state of hPSCs•Chemically defined medium reprograms hPSC lipogenic and redox metabolic pathways•hPSCs cultured in lipid-replete medium exhibit active oxidative mitochondrial flux•Glutamine fuels mitochondrial metabolism in self-renewing hPSCs
Zhang et al. apply metabolic flux analysis to comprehensively characterize the metabolism of human pluripotent stem cells cultured in different media. Cells maintained in chemically defined media significantly upregulate lipid biosynthesis and redox pathways to compensate for medium lipid deficiency while downregulating oxidative mitochondrial metabolism.
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal ...autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.
Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major ...histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy
. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB
, mutations that cause loss of MHC-I are rarely found
despite the frequent downregulation of MHC-I expression
. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8
T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive fibrosis and hypovascularization, resulting in significant intratumoral hypoxia (low oxygen) that contributes to its ...aggressiveness, therapeutic resistance, and high mortality. Despite oxygen being a fundamental requirement for many cellular and metabolic processes, and the severity of hypoxia in PDAC, the impact of oxygen deprivation on PDAC biology is poorly understood. Investigating how PDAC cells survive in the near absence of oxygen, we find that PDAC cell lines grow robustly in oxygen tensions down to 0.1%, maintaining mitochondrial morphology, membrane potential, and the oxidative metabolic activity required for the synthesis of key metabolites for proliferation. Disrupting electron transfer efficiency by targeting mitochondrial respiratory supercomplex assembly specifically affects hypoxic PDAC proliferation, metabolism, and in vivo tumor growth. Collectively, our results identify a mechanism that enables PDAC cells to thrive in severe, oxygen-limited microenvironments.
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•PDAC cells maintain proliferation and oxidative metabolism during severe hypoxia•Mitochondrial supercomplexes increase the metabolic efficiency of hypoxic PDAC cells•Inhibiting supercomplex formation impairs hypoxic PDAC growth and tumor formation•AOX expression rescues the growth defects observed upon loss of supercomplex formation
Pancreatic ductal adenocarcinoma (PDAC) is severely hypoxic to an extent predicted to have significant implications for the biology of these tumors. Hollinshead et al. demonstrate a role for mitochondrial respiratory supercomplexes in maintaining growth and oxidative mitochondrial metabolism in severely hypoxic pancreatic cancer cells.
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