bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2021‒03‒28
fifty-five papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit


  1. Nat Metab. 2021 Mar;3(3): 327-336
      Glycogen accumulation is a highly consistent, distinguishable characteristic of clear cell renal cell carcinoma (ccRCC)1. While elevated glycogen pools might be advantageous for ccRCC cells in nutrient-deprived microenvironments to sustain tumour viability, data supporting a biological role for glycogen in ccRCC are lacking. Here, we demonstrate that glycogen metabolism is not required for ccRCC proliferation in vitro nor xenograft tumour growth in vivo. Disruption of glycogen synthesis by CRISPR-mediated knockout of glycogen synthase 1 (GYS1) has no effect on proliferation in multiple cell lines, regardless of glucose concentrations or oxygen levels. Similarly, prevention of glycogen breakdown by deletion or pharmacological inhibition of glycogen phosphorylase B (PYGB) and L (PYGL) has no impact on cell viability under any condition tested. Lastly, in vivo xenograft experiments using the ccRCC cell line, UMRC2, reveal no substantial changes in tumour size or volume when glycogen metabolism is altered, largely mimicking the phenotype of our in vitro observations. Our findings suggest that glycogen build-up in established ccRCC tumour cells is likely to be a secondary, and apparently dispensable, consequence of constitutively active hypoxia-inducible factor 1-alpha (HIF-1α) signalling.
    DOI:  https://doi.org/10.1038/s42255-021-00367-x
  2. Nat Metab. 2021 Mar;3(3): 394-409
      Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid-induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.
    DOI:  https://doi.org/10.1038/s42255-021-00369-9
  3. Cell Metab. 2021 Mar 23. pii: S1550-4131(21)00110-8. [Epub ahead of print]
      Mitochondria have an independent genome (mtDNA) and protein synthesis machinery that coordinately activate for mitochondrial generation. Here, we report that the Krebs cycle intermediate fumarate links metabolism to mitobiogenesis through binding to malic enzyme 2 (ME2). Mechanistically, fumarate binds ME2 with two complementary consequences. First, promoting the formation of ME2 dimers, which activate deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT). DUT fosters thymidine generation and an increase of mtDNA. Second, fumarate-induced ME2 dimers abrogate ME2 monomer binding to mitochondrial ribosome protein L45, freeing it for mitoribosome assembly and mtDNA-encoded protein production. Methylation of the ME2-fumarate binding site by protein arginine methyltransferase-1 inhibits fumarate signaling to constrain mitobiogenesis. Notably, acute myeloid leukemia is highly dependent on mitochondrial function and is sensitive to targeting of the fumarate-ME2 axis. Therefore, mitobiogenesis can be manipulated in normal and malignant cells through ME2, an unanticipated governor of mitochondrial biomass production that senses nutrient availability through fumarate.
    Keywords:  acute myeloid leukemia; arginine methylation; deoxyuridine 5′-triphosphate nucleotidohydrolase; fumarate; malic enzyme 2; mitobiogenesis; mitochondrial ribosome; mitochondrial ribosome protein L45; protein arginine methyltransferase 1
    DOI:  https://doi.org/10.1016/j.cmet.2021.03.003
  4. Nat Commun. 2021 03 25. 12(1): 1876
      Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.
    DOI:  https://doi.org/10.1038/s41467-021-22166-4
  5. Mol Cell. 2021 Mar 19. pii: S1097-2765(21)00178-7. [Epub ahead of print]
      Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.
    Keywords:  MXD2; Protein translation; S6K1; WTAP; YTHDF readers; cMyc; eIF4A; m(6)A mRNA modification; mRNA stability; mTORC1
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.010
  6. Cancer Metab. 2021 Mar 24. 9(1): 13
      BACKGROUND: Majority of chondrosarcomas are associated with a number of genetic alterations, including somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, but the downstream effects of these mutated enzymes on cellular metabolism and tumor energetics are unknown. As IDH mutations are likely to be involved in malignant transformation of chondrosarcomas, we aimed to exploit metabolomic changes in IDH mutant and non-mutant chondrosarcomas.METHODS: Here, we profiled over 69 metabolites in 17 patient-derived xenografts by targeted mass spectrometry to determine if metabolomic differences exist in mutant IDH1, mutant IDH2, and non-mutant chondrosarcomas. UMAP (Uniform Manifold Approximation and Projection) analysis was performed on our dataset to examine potential similarities that may exist between each chondrosarcoma based on genotype.
    RESULTS: UMAP revealed that mutant IDH chondrosarcomas possess a distinct metabolic profile compared with non-mutant chondrosarcomas. More specifically, our targeted metabolomics study revealed large-scale differences in organic acid intermediates of the tricarboxylic acid (TCA) cycle, amino acids, and specific acylcarnitines in chondrosarcomas. Lactate and late TCA cycle intermediates were elevated in mutant IDH chondrosarcomas, suggestive of increased glycolytic metabolism and possible anaplerotic influx to the TCA cycle. A broad elevation of amino acids was found in mutant IDH chondrosarcomas. A few acylcarnitines of varying carbon chain lengths were also elevated in mutant IDH chondrosarcomas, but with minimal clustering in accordance with tumor genotype. Analysis of previously published gene expression profiling revealed increased expression of several metabolism genes in mutant IDH chondrosarcomas, which also correlated to patient survival.
    CONCLUSIONS: Overall, our findings suggest that IDH mutations induce global metabolic changes in chondrosarcomas and shed light on deranged metabolic pathways.
    Keywords:  Acylcarnitines; Amino acids; Cancer; Chondrosarcoma; Genetic mutation; Glycolysis; Metabolism; Mutant IDH; TCA cycle
    DOI:  https://doi.org/10.1186/s40170-021-00247-8
  7. Nat Metab. 2021 Mar;3(3): 410-427
      TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.
    DOI:  https://doi.org/10.1038/s42255-021-00368-w
  8. Nat Commun. 2021 Mar 26. 12(1): 1905
      Brown and beige adipose tissue are emerging as distinct endocrine organs. These tissues are functionally associated with skeletal muscle, adipose tissue metabolism and systemic energy expenditure, suggesting an interorgan signaling network. Using metabolomics, we identify 3-methyl-2-oxovaleric acid, 5-oxoproline, and β-hydroxyisobutyric acid as small molecule metabokines synthesized in browning adipocytes and secreted via monocarboxylate transporters. 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid induce a brown adipocyte-specific phenotype in white adipocytes and mitochondrial oxidative energy metabolism in skeletal myocytes both in vitro and in vivo. 3-methyl-2-oxovaleric acid and 5-oxoproline signal through cAMP-PKA-p38 MAPK and β-hydroxyisobutyric acid via mTOR. In humans, plasma and adipose tissue 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid concentrations correlate with markers of adipose browning and inversely associate with body mass index. These metabolites reduce adiposity, increase energy expenditure and improve glucose and insulin homeostasis in mouse models of obesity and diabetes. Our findings identify beige adipose-brown adipose-muscle physiological metabokine crosstalk.
    DOI:  https://doi.org/10.1038/s41467-021-22272-3
  9. Cell Rep. 2021 Mar 23. pii: S2211-1247(21)00189-3. [Epub ahead of print]34(12): 108875
      The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. Here, we show that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. Our data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations.
    Keywords:  ImpL2; PINK1; PTEN; Parkin; mitochondria
    DOI:  https://doi.org/10.1016/j.celrep.2021.108875
  10. Nat Commun. 2021 Mar 26. 12(1): 1929
      Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.
    DOI:  https://doi.org/10.1038/s41467-021-22117-z
  11. Nat Commun. 2021 Mar 26. 12(1): 1920
      Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.
    DOI:  https://doi.org/10.1038/s41467-021-22101-7
  12. Autophagy. 2021 Mar 23.
      Mitochondria are the main cellular energy powerhouses and supply most of the energy in the form of ATP to fuel essential neuronal functions through oxidative phosphorylation (OXPHOS). In Alzheimer disease (AD), metabolic and mitochondrial disruptions are an early feature preceding any histopathological and clinical manifestations. Mitochondrial malfunction is also linked to synaptic defects in early AD. Mitophagy serves as a key cellular quality control mechanism involving sequestration of damaged mitochondria within autophagosomes and their subsequent degradation in lysosomes. However, it remains largely unknown whether mitophagy is involved in the regulation of energy metabolism in neurons, and if so, whether metabolic deficiency in AD is attributed to mitophagy dysfunction. Here we reveal that mitophagy is broadly activated in metabolically enhanced neurons upon OXPHOS stimulation, which sustains high energetic activity by increasing mitochondrial turnover and hence facilitating mitochondrial maintenance. Unexpectedly, in AD-related mutant HsAPP Tg mouse brains, early stimulation of OXPHOS activity fails to correct energy deficits but exacerbates synapse loss as a consequence of mitophagy failure. Excitingly, lysosomal enhancement in AD neurons restores impaired metabolic function by promoting elimination of damaged mitochondria, protecting against synaptic damage in AD mouse brains. Taken together, we propose a new mechanism by which mitophagy controls bioenergetic status in neurons, furthering our understanding of the direct impact of mitophagy defects on AD-linked metabolic deficits and shedding light on the development of novel therapeutic strategies to treat AD by the early stimulation of mitochondrial metabolism combined with elevation of lysosomal proteolytic activity.
    Keywords:  Alzheimer; bioenergetics; energy metabolism; lysosomal proteolysis; metabolic deficiency; mitochondrial stress; mitophagosome; neuronal mitophagy; retrograde transport; synapse loss
    DOI:  https://doi.org/10.1080/15548627.2021.1907167
  13. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2100558118. [Epub ahead of print]118(13):
      Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c8-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.
    Keywords:  ATP synthase; TMEM242; TMEM70; assembly; human mitochondria
    DOI:  https://doi.org/10.1073/pnas.2100558118
  14. J Pathol. 2021 Mar 26.
      Glutamine is a critical nutrient in cancer, however its contribution to purine metabolism in prostate cancer has not previously been determined. Guanosine monophosphate synthetase (GMPS) acts in the de novo purine biosynthesis pathway, utilizing a glutamine amide to synthesize the guanine nucleotide. This study demonstrates that GMPS mRNA expression correlates with Gleason score in prostate cancer samples, while high GMPS expression was associated with decreased rates of overall and disease/progression-free survival. Pharmacological inhibition or knockdown of GMPS significantly decreased cell growth in both LNCaP and PC-3 prostate cancer cells. We utilized 15 N-(amide)-glutamine and U-13 C5 -glutamine metabolomics to dissect the pathways involved, and despite similar growth inhibition by GMPS knockdown, we show unique metabolic effects across each cell line. Using a PC-3 xenograft mouse model, tumor growth was also significantly decreased after GMPS knockdown, highlighting the importance of glutamine metabolism and providing support for GMPS as a therapeutic target in prostate cancer. This article is protected by copyright. All rights reserved.
    Keywords:  GMPS; cell growth; glutamine; immunohistochemistry; knockdown; metabolism; metabolomics; prostate cancer; purine; xenograft
    DOI:  https://doi.org/10.1002/path.5665
  15. Mol Cell. 2021 Mar 17. pii: S1097-2765(21)00177-5. [Epub ahead of print]
      The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N6-methyladenosine (m6A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m6A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m6A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m6A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth.
    Keywords:  Cell growth; MAT2A; Methionine cycle; N(6)-methyladenosine; Protein Synthesis; RNA metabolism; S-adenosylmethionine; WTAP; mTOR; mTORC1
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.009
  16. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2025383118. [Epub ahead of print]118(13):
      Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.
    Keywords:  MRI; biomarker; hyperpolarization; metabolism; parahydrogen
    DOI:  https://doi.org/10.1073/pnas.2025383118
  17. J Lipid Res. 2021 Mar 20. pii: S0022-2275(21)00051-1. [Epub ahead of print] 100069
      Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart, thus therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation deficient hearts due to carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine-shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability while cardiac and skeletal muscles depend on carnitine but not on CPT2.
    Keywords:  carnitine palmitoyltransferase; carnitine-shuttle; fatty acid oxidation; medium-chain fatty acids; mitochondria
    DOI:  https://doi.org/10.1016/j.jlr.2021.100069
  18. J Biochem Mol Toxicol. 2021 Mar 09. e22737
      Homocysteine (Hcy) is a sulfur-containing amino acid that originated in methionine metabolism and the elevated level of Hcy in plasma is considered to be an independent risk factor for cardiovascular diseases (CVD). Endothelial dysfunction plays a major role in the development of CVD, while the potential mechanism of Hcy-induced endothelial dysfunction is still unclear. Here, in Hcy-treated endothelial cells, we observed the destruction of mitochondrial morphology and the decline of mitochondrial membrane potential. Meanwhile, the level of ATP was reduced and the reactive oxygen species was increased. The expressions of dynamin-related protein 1 (Drp1) and phosphate-Drp1 (Ser616) were upregulated, whereas the expression of mitofusin 2 was inhibited by Hcy treatment. These findings suggested that Hcy not only triggered mitochondrial dysfunction but also incurred an imbalance of mitochondrial dynamics in endothelial cells. The expression of mitochondrial calcium uniporter (MCU) was activated by Hcy, contributing to calcium transferring into mitochondria. Interestingly, the formation of mitochondria-associated membranes (MAMs) was increased in endothelial cells after Hcy administration. The inositol 1,4,5-triphosphate receptor (IP3R)-glucose-regulated protein 75 (Grp75)-voltage-dependent anion channel (VDAC) complex, which was enriched in MAMs, was also increased. The accumulation of mitochondrial calcium could be blocked by inhibiting with the IP3R inhibitor Xestospongin C (XeC) in Hcy-treated cells. Then, we confirmed that the mitochondrial dysfunction and the increased mitochondrial fission induced by Hcy could be attenuated after Hcy and XeC co-treatment. In conclusion, Hcy-induced mitochondrial dysfunction and dynamics disorder in endothelial cells were mainly related to the increase of calcium as a result of the upregulated expressions of the MCU and the IP3R-Grp75-VDAC complex in MAMs.
    Keywords:  calcium; endothelial cells; homocysteine; mitochondria-associated membranes; mitochondrial dysfunction
    DOI:  https://doi.org/10.1002/jbt.22737
  19. BMC Genomics. 2021 Mar 24. 22(1): 213
      BACKGROUND: In addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood.RESULTS: Using a Drosophila model in which alternative mtDNAs are present on a common nuclear background, we studied the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status. Adult flies with the 'native' and 'disrupted' genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment.
    CONCLUSIONS: Many of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.
    Keywords:  Mitochondrial introgression; Mitonuclear genotype; Rapamycin; mTORC1
    DOI:  https://doi.org/10.1186/s12864-021-07516-2
  20. PLoS One. 2021 ;16(3): e0249047
      Mitochondria are commonly viewed as highly elongated organelles with regularly spaced mtDNA genomes organized as compact nucleoids that generate the local transcripts essential for production of mitochondrial ribosomes and key components of the respiratory chain. In contrast, A549 human lung carcinoma cells frequently contain apparently swollen mitochondria harboring multiple discrete mtDNA nucleoids and RNA processing granules in a contiguous matrix compartment. While this seemingly aberrant mitochondrial morphology is akin to "mito-bulbs" previously described in cells exposed to a variety of genomic stressors, it occurs in A549 cells under typical culture conditions. We provide a detailed confocal and super-resolution microscopic investigation of the incidence of such mito-bulbs in A549 cells. Most mito-bulbs appear stable, engage in active replication and transcription, and maintain respiration but feature an elevated oxidative environment. High concentrations of glucose and/or L-glutamine in growth media promote a greater incidence of mito-bulbs. Furthermore, we demonstrate that treatment of A549 cells with TGFβ suppresses the formation of mito-bulbs while treatment with a specific TGFβ pathway inhibitor substantially increases incidence. This striking heterogeneity of mitochondrial form and function may play an important role in a variety of diseases involving mitochondrial dysfunction.
    DOI:  https://doi.org/10.1371/journal.pone.0249047
  21. iScience. 2021 Mar 19. 24(3): 102217
      Systemic metabolic homeostasis is regulated by inter-organ metabolic cycles involving multiple organs. Obesity impairs inter-organ metabolic cycles, resulting in metabolic diseases. The systemic landscape of dysregulated inter-organ metabolic cycles in obesity has yet to be explored. Here, we measured the transcriptome, proteome, and metabolome in the liver and skeletal muscle and the metabolome in blood of fasted wild-type and leptin-deficient obese (ob/ob) mice, identifying components with differential abundance and differential regulation in ob/ob mice. By constructing and evaluating the trans-omic network controlling the differences in metabolic reactions between fasted wild-type and ob/ob mice, we provided potential mechanisms of the obesity-associated dysfunctions of metabolic cycles between liver and skeletal muscle involving glucose-alanine, glucose-lactate, and ketone bodies. Our study revealed obesity-associated systemic pathological mechanisms of dysfunction of inter-organ metabolic cycles.
    Keywords:  Biological Sciences; Endocrinology; Metabolomics; Omics; Proteomics; Systems Biology; Transcriptomic
    DOI:  https://doi.org/10.1016/j.isci.2021.102217
  22. BMC Cancer. 2021 Mar 24. 21(1): 312
      BACKGROUND: Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported.METHODS: Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database.
    RESULTS: FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation.
    CONCLUSIONS: We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.
    Keywords:  Cell cycle; DNA damage; Forkhead; Kidney cancer
    DOI:  https://doi.org/10.1186/s12885-021-07957-8
  23. Nature. 2021 Mar 25.
      Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
    DOI:  https://doi.org/10.1038/s41586-021-03468-5
  24. BMC Biol. 2021 Mar 24. 19(1): 57
      BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential.RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein.
    CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
    Keywords:  High-throughput screen; Luteolin; Mitochondria; Mitochondria-ER contacts; Mitochondrial calcium
    DOI:  https://doi.org/10.1186/s12915-021-00979-5
  25. Br J Cancer. 2021 Mar 25.
      Classification of cancer should lead to informative patients' stratification and selective therapeutic vulnerabilities. A pathway-based classification of glioblastoma uncovered a mitochondrial subtype with a unique sensitivity to inhibitors of oxidative phosphorylation. Precision targeting of cancer metabolism could provide therapeutic opportunities to a lethal neoplasm and be translated to other tumour types.
    DOI:  https://doi.org/10.1038/s41416-021-01360-7
  26. Cell Death Dis. 2021 Mar 25. 12(4): 318
      A growing number of studies supports the existence of a dynamic interplay between energetic metabolism and autophagy, whose induction represents an adaptive response against several stress conditions. Autophagy is an evolutionarily conserved and a highly orchestrated catabolic recycling process that guarantees cellular homeostasis. To date, the exact role of autophagy in vitiligo pathogenesis is still not clear. Here, we provide the first evidence that autophagy occurs in melanocytes and fibroblasts from non-lesional skin of vitiligo patients, as a result of metabolic surveillance response. More precisely, this study is the first to reveal that induction of autophagy exerts a protective role against the intrinsic metabolic stress and attempts to antagonize degenerative processes in normal appearing vitiligo skin, where melanocytes and fibroblasts are already prone to premature senescence.
    DOI:  https://doi.org/10.1038/s41419-021-03592-0
  27. Anal Chim Acta. 2021 Apr 22. pii: S0003-2670(21)00168-9. [Epub ahead of print]1155 338342
      Spatially resolved metabolomics offers unprecedented opportunities for elucidating metabolic mechanisms during cancer progression. It facilitated the discovery of aberrant cellular metabolism with clinical application potential. Here, we developed a novel strategy to discover cancer tissue relevant metabolic signatures by high spatially resolved metabolomics combined with a multicellular tumor spheroid (MCTS) in vitro model. Esophageal cancer MCTS were generated using KYSE-30 human esophageal cancer cells to fully mimic the 3D microenvironment under physiological conditions. Then, the spatial features and temporal variation of metabolites and metabolic pathways in MCTS were accurately mapped by using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with a spatial resolution at ∼12 μm. Metabolites, such as glutamate, tyrosine, inosine and various types of lipids displayed heterogeneous distributions in different microregions inside the MCTS, revealing the metabolic heterogenicity of cancer cells under different proliferative states. Subsequently, through joint analysis of metabolomic data of clinical cancer tissue samples, cancer tissue relevant metabolic signatures in esophageal cancer MCTS were identified, including glutamine metabolism, fatty acid metabolism, de novo synthesis phosphatidylcholine (PC) and phosphatidylethanolamine (PE), etc. In addition, the abnormal expression of the involved metabolic enzymes, i.e., GLS, FASN, CHKA and cPLA2, was further confirmed and showed similar tendencies in esophageal cancer MCTS and cancer tissues. The MALDI-MSI combined with MCTS approach offers molecular insights into cancer metabolism with real-word relevance, which would potentially benefit the biomarker discovery and metabolic mechanism studies.
    Keywords:  Cancer metabolism; Esophageal cancer; Mass spectrometry imaging; Multicellular tumor spheroids; Spatially resolved metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2021.338342
  28. Mitochondrion. 2021 Mar 17. pii: S1567-7249(21)00035-0. [Epub ahead of print]
      Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.
    Keywords:  ATP synthesis; CRISPR/Cas9; Complex I; Electron transport chain; ROS; apoptosis; glycolysis; idebenone; necrosis; oxygen consumption; respiration
    DOI:  https://doi.org/10.1016/j.mito.2021.03.003
  29. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2008772118. [Epub ahead of print]118(13):
      Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.
    Keywords:  CRISPR; TERT; cancer; glioblastoma; temozolomide
    DOI:  https://doi.org/10.1073/pnas.2008772118
  30. Nat Biotechnol. 2021 Mar 25.
      Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.
    DOI:  https://doi.org/10.1038/s41587-021-00860-4
  31. EMBO Rep. 2021 Mar 22. e50766
      SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.
    Keywords:  kidney tubule; renal tubular acidosis; sirtuins
    DOI:  https://doi.org/10.15252/embr.202050766
  32. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2012748118. [Epub ahead of print]118(13):
      Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.
    Keywords:  GAC; glutaminase; prostate cancer; therapeutic resistance
    DOI:  https://doi.org/10.1073/pnas.2012748118
  33. Comp Biochem Physiol B Biochem Mol Biol. 2021 Mar 20. pii: S1096-4959(21)00035-X. [Epub ahead of print]255 110596
      Oxidative phosphorylation is compromised in hypoxia, but many organisms live and exercise in low oxygen environments. Hypoxia-driven adaptations at the mitochondrial level are common and may enhance energetic efficiency or minimize deleterious reactive oxygen species (ROS) generation. Mitochondria from various hypoxia-tolerant animals exhibit robust functional changes following in vivo hypoxia and we hypothesized that similar plasticity would occur in naked mole-rat skeletal muscle. To test this, we exposed adult subordinate naked mole-rats to normoxia (21% O2) or acute (4 h, 7% O2) or chronic hypoxia (4-6 weeks, 11% O2) and then isolated skeletal muscle mitochondria. Using high-resolution respirometry and a fluorescent indicator of ROS production, we then probed for changes in: i) lipid- (palmitoylcarnitine-malate), ii) carbohydrate- (pyruvate-malate), and iii) succinate-fueled metabolism, and also iv) complex IV electron transfer capacity, and v) H2O2 production. Compared to normoxic values, a) lipid-fueled uncoupled respiration was reduced ~15% during acute and chronic hypoxia, b) complex I-II capacity and the rate of ROS efflux were both unaffected, and c) complex II and IV uncoupled respiration were supressed ~16% following acute hypoxia. Notably, complex II-linked H2O2 efflux was 33% lower after acute hypoxia, which may reduce deleterious ROS bursts during reoxygenation. These mild changes in lipid- and carbohydrate-fueled respiratory capacity may reflect the need for this animal to exercise regularly in highly variable and intermittently hypoxic environments in which more robust plasticity may be energetically expensive.
    Keywords:  Electron transport system; High resolution respirometry; Oxidative phosphorylation; Reactive oxygen species; Succinate
    DOI:  https://doi.org/10.1016/j.cbpb.2021.110596
  34. Br J Cancer. 2021 Mar 25.
      Recent advances in our understanding of tumour heterogeneity alongside studies investigating altered metabolism within transformed tissue have identified metabolic pathways critical to cancer cell survival. Leveraging this information presents a promising new avenue for the generation of cancer-specific therapeutics and improved patient outcomes.
    DOI:  https://doi.org/10.1038/s41416-021-01345-6
  35. Commun Biol. 2021 Mar 26. 4(1): 417
      Tumour evolution is driven by both genetic and epigenetic changes. CENP-A, the centromeric histone H3 variant, is an epigenetic mark that directly perturbs genetic stability and chromatin when overexpressed. Although CENP-A overexpression is a common feature of many cancers, how this impacts cell fate and response to therapy remains unclear. Here, we established a tunable system of inducible and reversible CENP-A overexpression combined with a switch in p53 status in human cell lines. Through clonogenic survival assays, single-cell RNA-sequencing and cell trajectory analysis, we uncover the tumour suppressor p53 as a key determinant of how CENP-A impacts cell state, cell identity and therapeutic response. If p53 is functional, CENP-A overexpression promotes senescence and radiosensitivity. Surprisingly, when we inactivate p53, CENP-A overexpression instead promotes epithelial-mesenchymal transition, an essential process in mammalian development but also a precursor for tumour cell invasion and metastasis. Thus, we uncover an unanticipated function of CENP-A overexpression to promote cell fate reprogramming, with important implications for development and tumour evolution.
    DOI:  https://doi.org/10.1038/s42003-021-01941-5
  36. Mol Genet Metab. 2021 Mar 11. pii: S1096-7192(21)00062-7. [Epub ahead of print]
      Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.
    Keywords:  A-V difference; Arterial-venous difference; Arteriovenous difference; Brain; Hypoxia; Leigh syndrome; Metabolism; Metabolomics; NAD; Ndufs4; Niacin; Nicotinamide adenine dinucleotide; Nicotinic acid; O(2); Oxygen
    DOI:  https://doi.org/10.1016/j.ymgme.2021.03.005
  37. Free Radic Biol Med. 2021 Mar 19. pii: S0891-5849(21)00163-5. [Epub ahead of print]
      Coenzyme Q (CoQ) is ubiquitously embedded in lipid bilayers of various cellular organelles. As a redox cycler, CoQ shuttles electrons between mitochondrial complexes and extramitochondrial reductases and oxidases. In this way, CoQ is crucial for maintaining the mitochondrial function, ATP synthesis, and redox homeostasis. Cardiomyocytes have a high metabolic rate and rely heavily on mitochondria to provide energy. CoQ levels, in both plasma and the heart, correlate with heart failure in patients, indicating that CoQ is critical for cardiac function. Moreover, CoQ supplementation in clinics showed promising results for treating heart failure. This review provides a comprehensive view of CoQ metabolism and its interaction with redox enzymes and reactive species. We summarize the clinical trials and applications of CoQ in heart failure and discuss the caveats and future directions to improve CoQ therapeutics.
    Keywords:  CoQ; heart failure; redox
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.03.011
  38. Theranostics. 2021 ;11(9): 4011-4029
      Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro, ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.
    Keywords:  ADSL; colorectal cancer; fumarate; mTOR-MYC-axis; mitochondria
    DOI:  https://doi.org/10.7150/thno.50051
  39. Nat Commun. 2021 03 22. 12(1): 1812
      Human hexokinase 2 is an essential regulator of glycolysis that couples metabolic and proliferative activities in cancer cells. The binding of hexokinase 2 to the outer membrane of mitochondria is critical for its oncogenic activity. However, the regulation of hexokinase 2 binding to mitochondria remains unclear. Here, we report that SUMOylation regulates the binding of hexokinase 2 to mitochondria. We find that hexokinase 2 can be SUMOylated at K315 and K492. SUMO-specific protease SENP1 mediates the de-SUMOylation of hexokinase 2. SUMO-defective hexokinase 2 preferably binds to mitochondria and enhances both glucose consumption and lactate production and decreases mitochondrial respiration in parallel. This metabolic reprogramming supports prostate cancer cell proliferation and protects cells from chemotherapy-induced cell apoptosis. Moreover, we demonstrate an inverse relationship between SENP1-hexokinase 2 axis and chemotherapy response in prostate cancer samples. Our data provide evidence for a previously uncovered posttranslational modification of hexokinase 2 in cancer cells, suggesting a potentially actionable strategy for preventing chemotherapy resistance in prostate cancer.
    DOI:  https://doi.org/10.1038/s41467-021-22163-7
  40. Cell Mol Life Sci. 2021 Mar 22.
      The remodeling of the mitochondrial network is a critical process in maintaining cellular homeostasis and is intimately related to mitochondrial function. The interplay between the formation of new mitochondria (biogenesis) and the removal of damaged mitochondria (mitophagy) provide a means for the repopulation of the mitochondrial network. Additionally, mitochondrial fission and fusion serve as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been characterised in multiple tissue- and cell-types, and under various conditions. In skeletal muscle, the robust remodeling of the mitochondrial network is observed, particularly after injury where large portions of the tissue/cell structures are damaged. The significance of mitochondrial remodeling in regulating skeletal muscle regeneration has been widely studied, with alterations in mitochondrial remodeling processes leading to incomplete regeneration and impaired skeletal muscle function. Needless to say, important questions related to mitochondrial remodeling and skeletal muscle regeneration still remain unanswered and require further investigation. Therefore, this review will discuss the known molecular mechanisms of mitochondrial network remodeling, as well as integrate these mechanisms and discuss their relevance in myogenesis and regenerating skeletal muscle.
    Keywords:  Biogenesis; Fission; Fusion; Mitochondria; Mitophagy; Regeneration; Skeletal muscle; Skeletal muscle stem cells
    DOI:  https://doi.org/10.1007/s00018-021-03807-9
  41. Br J Cancer. 2021 Mar 25.
      BACKGROUND: Recent studies have emphasised the important role of amino acids in cancer metabolism. Cold physical plasma is an evolving technology employed to target tumour cells by introducing reactive oxygen species (ROS). However, limited understanding is available on the role of metabolic reprogramming in tumour cells fostering or reducing plasma-induced cancer cell death.METHODS: The utilisation and impact of major metabolic substrates of fatty acid, amino acid and TCA pathways were investigated in several tumour cell lines following plasma exposure by qPCR, immunoblotting and cell death analysis.
    RESULTS: Metabolic substrates were utilised in Panc-1 and HeLa but not in OVCAR3 and SK-MEL-28 cells following plasma treatment. Among the key genes governing these pathways, ASCT2 and SLC3A2 were consistently upregulated in Panc-1, Miapaca2GR, HeLa and MeWo cells. siRNA-mediated knockdown of ASCT2, glutamine depletion and pharmacological inhibition with V9302 sensitised HeLa cells to the plasma-induced cell death. Exogenous supplementation of glutamine, valine or tyrosine led to improved metabolism and viability of tumour cells following plasma treatment.
    CONCLUSION: These data suggest the amino acid influx driving metabolic reprogramming in tumour cells exposed to physical plasma, governing the extent of cell death. This pathway could be targeted in combination with existing anti-tumour agents.
    DOI:  https://doi.org/10.1038/s41416-021-01335-8
  42. Nat Commun. 2021 03 22. 12(1): 1808
      Mutational activation of KRAS promotes the initiation and progression of cancers, especially in the colorectum, pancreas, lung, and blood plasma, with varying prevalence of specific activating missense mutations. Although epidemiological studies connect specific alleles to clinical outcomes, the mechanisms underlying the distinct clinical characteristics of mutant KRAS alleles are unclear. Here, we analyze 13,492 samples from these four tumor types to examine allele- and tissue-specific genetic properties associated with oncogenic KRAS mutations. The prevalence of known mutagenic mechanisms partially explains the observed spectrum of KRAS activating mutations. However, there are substantial differences between the observed and predicted frequencies for many alleles, suggesting that biological selection underlies the tissue-specific frequencies of mutant alleles. Consistent with experimental studies that have identified distinct signaling properties associated with each mutant form of KRAS, our genetic analysis reveals that each KRAS allele is associated with a distinct tissue-specific comutation network. Moreover, we identify tissue-specific genetic dependencies associated with specific mutant KRAS alleles. Overall, this analysis demonstrates that the genetic interactions of oncogenic KRAS mutations are allele- and tissue-specific, underscoring the complexity that drives their clinical consequences.
    DOI:  https://doi.org/10.1038/s41467-021-22125-z
  43. Nat Rev Mol Cell Biol. 2021 Mar 23.
      Brown and beige adipocytes are mitochondria-enriched cells capable of dissipating energy in the form of heat. These thermogenic fat cells were originally considered to function solely in heat generation through the action of the mitochondrial protein uncoupling protein 1 (UCP1). In recent years, significant advances have been made in our understanding of the ontogeny, bioenergetics and physiological functions of thermogenic fat. Distinct subtypes of thermogenic adipocytes have been identified with unique developmental origins, which have been increasingly dissected in cellular and molecular detail. Moreover, several UCP1-independent thermogenic mechanisms have been described, expanding the role of these cells in energy homeostasis. Recent studies have also delineated roles for these cells beyond the regulation of thermogenesis, including as dynamic secretory cells and as a metabolic sink. This Review presents our current understanding of thermogenic adipocytes with an emphasis on their development, biological functions and roles in systemic physiology.
    DOI:  https://doi.org/10.1038/s41580-021-00350-0
  44. Nat Metab. 2021 Mar;3(3): 366-377
      Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.
    DOI:  https://doi.org/10.1038/s42255-021-00361-3
  45. Nature. 2021 Mar 24.
      Our knowledge of copy number evolution during the expansion of primary breast tumours is limited1,2. Here, to investigate this process, we developed a single-cell, single-molecule DNA-sequencing method and performed copy number analysis of 16,178 single cells from 8 human triple-negative breast cancers and 4 cell lines. The results show that breast tumours and cell lines comprise a large milieu of subclones (7-22) that are organized into a few (3-5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple loss-of-heterozygosity events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumour expansion. By subcloning single daughter cells in culture, we show that tumour cells rediversify their genomes and do not retain isogenic properties. These data show that triple-negative breast cancers continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumour growth.
    DOI:  https://doi.org/10.1038/s41586-021-03357-x
  46. Life Sci Alliance. 2021 06;pii: e202000903. [Epub ahead of print]4(6):
      Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.
    DOI:  https://doi.org/10.26508/lsa.202000903
  47. Cancer Res. 2021 Mar 24. pii: canres.3134.2020. [Epub ahead of print]
      Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3134
  48. Nat Rev Nephrol. 2021 Mar 25.
      Complex multicellular life in mammals relies on functional cooperation of different organs for the survival of the whole organism. The kidneys play a critical part in this process through the maintenance of fluid volume and composition homeostasis, which enables other organs to fulfil their tasks. The renal endothelium exhibits phenotypic and molecular traits that distinguish it from endothelia of other organs. Moreover, the adult kidney vasculature comprises diverse populations of mostly quiescent, but not metabolically inactive, endothelial cells (ECs) that reside within the kidney glomeruli, cortex and medulla. Each of these populations supports specific functions, for example, in the filtration of blood plasma, the reabsorption and secretion of water and solutes, and the concentration of urine. Transcriptional profiling of these diverse EC populations suggests they have adapted to local microenvironmental conditions (hypoxia, shear stress, hyperosmolarity), enabling them to support kidney functions. Exposure of ECs to microenvironment-derived angiogenic factors affects their metabolism, and sustains kidney development and homeostasis, whereas EC-derived angiocrine factors preserve distinct microenvironment niches. In the context of kidney disease, renal ECs show alteration in their metabolism and phenotype in response to pathological changes in the local microenvironment, further promoting kidney dysfunction. Understanding the diversity and specialization of kidney ECs could provide new avenues for the treatment of kidney diseases and kidney regeneration.
    DOI:  https://doi.org/10.1038/s41581-021-00411-9
  49. Dev Cell. 2021 Mar 22. pii: S1534-5807(21)00207-0. [Epub ahead of print]56(6): 721-722
      In this issue of Developmental Cell,Yang et al. (2021) discover that, RHEB traffics to mitochondria to promote energy production by stimulating pyruvate dehydrogenase to convert pyruvate to acetyl-CoA.
    DOI:  https://doi.org/10.1016/j.devcel.2021.03.010
  50. Cancer Metab. 2021 Mar 24. 9(1): 12
      BACKGROUND: Fructose is an abundant source of carbon and energy for cells to use for metabolism, but only certain cell types use fructose to proliferate. Tumor cells that acquire the ability to metabolize fructose have a fitness advantage over their neighboring cells, but the proteins that mediate fructose metabolism in this context are unknown. Here, we investigated the determinants of fructose-mediated cell proliferation.METHODS: Live cell imaging and crystal violet assays were used to characterize the ability of several cell lines (RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, PC3, DLD1 HCT116, and 22RV1) to proliferate in fructose (i.e., the fructolytic ability). Fructose metabolism gene expression was determined by RT-qPCR and western blot for each cell line. A positive selection approach was used to "train" non-fructolytic PC3 cells to utilize fructose for proliferation. RNA-seq was performed on parental and trained PC3 cells to find key transcripts associated with fructolytic ability. A CRISPR-cas9 plasmid containing KHK-specific sgRNA was transfected in 293T cells to generate KHK-/- cells. Lentiviral transduction was used to overexpress empty vector, KHK, or GLUT5 in cells. Metabolic profiling was done with seahorse metabolic flux analysis as well as LC/MS metabolomics. Cell Titer Glo was used to determine cell sensitivity to 2-deoxyglucose in media containing either fructose or glucose.
    RESULTS: We found that neither the tissue of origin nor expression level of any single gene related to fructose catabolism determine the fructolytic ability. However, cells cultured chronically in fructose can develop fructolytic ability. SLC2A5, encoding the fructose transporter, GLUT5, was specifically upregulated in these cells. Overexpression of GLUT5 in non-fructolytic cells enabled growth in fructose-containing media across cells of different origins. GLUT5 permitted fructose to flux through glycolysis using hexokinase (HK) and not ketohexokinase (KHK).
    CONCLUSIONS: We show that GLUT5 is a robust and generalizable driver of fructose-dependent cell proliferation. This indicates that fructose uptake is the limiting factor for fructose-mediated cell proliferation. We further demonstrate that cellular proliferation with fructose is independent of KHK.
    Keywords:  Fructose; GLUT5 (SLC2A5); Hexokinase; Ketohexokinase; Metabolism
    DOI:  https://doi.org/10.1186/s40170-021-00246-9
  51. Nat Rev Cancer. 2021 Mar 23.
      Autophagy is a regulated mechanism that removes unnecessary or dysfunctional cellular components and recycles metabolic substrates. In response to stress signals in the tumour microenvironment, the autophagy pathway is altered in tumour cells and immune cells - thereby differentially affecting tumour progression, immunity and therapy. In this Review, we summarize our current understanding of the immunologically associated roles and modes of action of the autophagy pathway in cancer progression and therapy, and discuss potential approaches targeting autophagy to enhance antitumour immunity and improve the efficacy of current cancer therapy.
    DOI:  https://doi.org/10.1038/s41568-021-00344-2
  52. Metab Eng. 2021 Mar 23. pii: S1096-7176(21)00049-5. [Epub ahead of print]
      Genome-scale metabolic models describe cellular metabolism with mechanistic detail. Given their high complexity, such models need to be parameterized correctly to yield accurate predictions and avoid overfitting. Effective parameterization has been well-studied for microbial models, but it remains unclear for higher eukaryotes, including mammalian cells. To address this, we enumerated model parameters that describe key features of cultured mammalian cells - including cellular composition, bioprocess performance metrics, mammalian-specific pathways, and biological assumptions behind model formulation approaches. We tested these parameters by building thousands of metabolic models and evaluating their ability to predict the growth rates of a panel of phenotypically diverse Chinese Hamster Ovary cell clones. We found the following considerations to be most critical for accurate parameterization: (1) cells limit metabolic activity to maintain homeostasis, (2) cell morphology and viability change dynamically during a growth curve, and (3) cellular biomass has a particular macromolecular composition. Depending on parameterization, models predicted different metabolic phenotypes, including contrasting mechanisms of nutrient utilization and energy generation, leading to varying accuracies of growth rate predictions. Notably, accurate parameter values broadly agreed with experimental measurements. These insights will guide future investigations of mammalian metabolism.
    Keywords:  Bioprocess; Chinese Hamster Ovary cells; Flux Balance Analysis; Metabolic Network Modeling
    DOI:  https://doi.org/10.1016/j.ymben.2021.03.013
  53. Cancer Res. 2021 Mar 24. pii: canres.3730.2020. [Epub ahead of print]
      Oncometabolites are pathognomonic hallmarks in human cancers, including glioma, leukemia, neuroendocrine tumors, and renal cancer. Oncometabolites are aberrantly accumulated from disrupted Krebs cycle and affect the catalytic activity of α-ketoglutarate-dependent dioxygenases. Oncometabolites indicate distinct cancer-related patterns ranging from oncogenesis and metabolism to therapeutic resistance. Here we discuss the current understanding of oncometabolites as well as the controversies and challenges associated with oncometabolite-driven cancers. New insights into the relationship between cancer and oncometabolites will elucidate novel therapeutic avenues for improved cancer treatment.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3730