bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒08‒30
fifty-six papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31044-5. [Epub ahead of print]32(8): 108059
      The proteasome is the main proteolytic system for targeted protein degradation in the cell and is fine-tuned according to cellular needs. Here, we demonstrate that mitochondrial dysfunction and concomitant metabolic reprogramming of the tricarboxylic acid (TCA) cycle reduce the assembly and activity of the 26S proteasome. Both mitochondrial mutations in respiratory complex I and treatment with the anti-diabetic drug metformin impair 26S proteasome activity. Defective 26S assembly is reversible and can be overcome by supplementation of aspartate or pyruvate. This metabolic regulation of 26S activity involves specific regulation of proteasome assembly factors via the mTORC1 pathway. Of note, reducing 26S activity by metformin confers increased resistance toward the proteasome inhibitor bortezomib, which is reversible upon pyruvate supplementation. Our study uncovers unexpected consequences of defective mitochondrial metabolism for proteasomal protein degradation in the cell, which has important pathophysiological and therapeutic implications.
    Keywords:  26S proteasome; Rpn6; TCA; aspartate; metabolic reprogramming; metformin; mitochondria; proteasome assembly factors; proteasome inhibitor resistance; pyruvate; respiratory complex I
  2. Redox Biol. 2020 Aug 13. pii: S2213-2317(20)30890-9. [Epub ahead of print]36 101685
      NADPH is a pivotal cofactor that maintains redox homeostasis and lipogenesis in cancer cells and interference with NADPH production is a promising approach for treating cancer. However, how normal and cancer cells differentially exploit NADPH-producing pathways is unclear, and selective approaches to targeting NADPH are lacking. Here, we show that the assayed cancer cell lines preferentially depend on ME1-mediated NADPH production. ME1 knockdown increases intracellular ROS levels and impairs lipogenesis in cancer cells, leading to retarded proliferation and increased anoikis, while sparing normal cells. Notably, ME1 interference ultimately resulted in adaptive upregulation of mitochondrial IDH2 dependent of AMPK-FoxO1 activation to replenish the NADPH pool and mitigate cytosolic ROS. Combining ME1 ablation and IDH2 inhibition drastically reduces intracellular NADPH and prevents resistance to ME1 interference, resulting in increased apoptosis and impeded tumor growth and metastasis. This study demonstrates that cytosolic ME1 integrated with mitochondrial IDH2 is essential for tumor growth and metastasis, thereby highlighting the blockade of metabolic compensation by disrupting mitochondrial-cytosol NADPH transport as a promising approach to selectively targeting NADPH in cancer cells that rely on NADPH-driven antioxidant systems.
    Keywords:  IDH2; ME1; Metabolic adaptability; NADPH; ROS; Reductive carboxylation
  3. Cancers (Basel). 2020 Aug 24. pii: E2400. [Epub ahead of print]12(9):
      Targeting oxidative phosphorylation (OXPHOS) is a promising strategy to improve treatment outcomes of acute myeloid leukemia (AML) patients. IACS-010759 is a mitochondrial complex I inhibitor that has demonstrated preclinical antileukemic activity and is being tested in Phase I clinical trials. However, complex I deficiency has been reported to inhibit apoptotic cell death through prevention of cytochrome c release. Thus, combining IACS-010759 with a BH3 mimetic may overcome this mechanism of resistance leading to synergistic antileukemic activity against AML. In this study, we show that IACS-010759 and venetoclax synergistically induce apoptosis in OXPHOS-reliant AML cell lines and primary patient samples and cooperatively target leukemia progenitor cells. In a relatively OXPHOS-reliant AML cell line derived xenograft mouse model, IACS-010759 treatment significantly prolonged survival, which was further enhanced by treatment with IACS-010759 in combination with venetoclax. Consistent with our hypothesis, IACS-010759 treatment indeed retained cytochrome c in mitochondria, which was completely abolished by venetoclax, resulting in Bak/Bax- and caspase-dependent apoptosis. Our preclinical data provide a rationale for further development of the combination of IACS-010759 and venetoclax for the treatment of patients with AML.
    Keywords:  Bcl-2; IACS-010759; acute myeloid leukemia; oxidative phosphorylation; venetoclax
  4. Front Oncol. 2020 ;10 1217
      Esophageal squamous cell carcinoma (ESCC) has a worldwide impact on human health, due to its high incidence and mortality. Therefore, identifying compounds to increase patients' survival rate is urgently needed. Mefloquine (MQ) is an FDA-approved anti-malarial drug, which has been reported to inhibit cellular proliferation in several cancers. However, the anti-tumor activities of the drug have not yet been completely defined. In this study, mass spectrometry was employed to profile proteome changes in ESCC cells after MQ treatment. Sub-cellular localization and gene ontology term enrichment analysis suggested that MQ treatment mainly affect mitochondria. The KEGG pathway enrichment map of down-regulated pathways and Venn diagram indicated that all of the top five down regulated signaling pathways contain four key mitochondrial proteins (succinate dehydrogenase complex subunit C (SDHC), succinate dehydrogenase complex subunit D, mitochondrially encoded cytochrome c oxidase III and NADH: ubiquinone oxidoreductase subunit V3). Meanwhile, mitochondrial autophagy was observed in MQ-treated KYSE150 cells. More importantly, patient-derived xenograft mouse models of ESCC with SDHC high expression were more sensitive to MQ treatment than low SDHC-expressing xenografts. Taken together, mefloquine inhibits ESCC tumor growth by inducing mitochondrial autophagy and SDHC plays a vital role in MQ-induced anti-tumor effect on ESCC.
    Keywords:  autophagy; esophageal squamous cell carcinoma (ESCC); mefloquine (MQ); mitochondria; succinate dehydrogenase complex subunit C (SDHC)
  5. Am J Physiol Cell Physiol. 2020 Aug 26.
      Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high-glucose (15.6 mM) and high-fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration and energy expenditure were examined in obese resistant (OR), and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10-weeks with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.
    Keywords:  fission; fusion; mitochondria; obesity; respiration
  6. Sci Rep. 2020 Aug 25. 10(1): 14174
      Mitochondrial dysfunction and significant changes in metabolic pathways accompany cancer development and are responsible for maintaining the tumor microenvironment. Normal mitochondria can trigger intrinsic apoptosis by releasing cytochrome c into the cytosol. The survival of malignant cells highly depends on the suppression of this function. We validated that A250, a highly purified fraction of fermented wheat germ extract (FWGE), increases the carbon flux into the mitochondria, the expression of key elements of the Krebs cycle and oxidative phosphorylation (OXPHOS). The increased respiratory chain activity is related to the mitochondria's ability to release cytochrome c into the cytosol, which triggers the apoptotic cascade. The 68% tumor growth inhibitory effect observed in the murine melanoma study is related to this effect, as proteomic analysis validated similar changes in mitochondrial protein levels in the isolated tumor tissue samples. Blood count data indicated that this effect was not accompanied by general toxicity. This study is significant, as it shows that a highly concentrated form of FWGE is an effective agent that increases normal mitochondrial functionality. The lack of hepatotoxic and general toxic effects makes A250 an excellent candidate targeting mitochondria function in cancer therapy.
  7. Front Immunol. 2020 ;11 1968
      Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition.
    Keywords:  ATP citrate lyase; MHC class I chain-related proteins A; Natural Killer Group 2D; cancer metabolism; citrate; tumor immunology
  8. Nat Commun. 2020 Aug 28. 11(1): 4330
      Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.
  9. FASEB J. 2020 Aug 26.
      The zinc-finger protein ZBTB20 regulates development and metabolism in multiple systems, and is essential for postnatal survival in mice. However, its potential role in the cardiovascular system remains undefined. Here, we demonstrate that ZBTB20 is critically involved in the regulation of cardiac contractility and blood pressure in mice. At the age of 16 days, the relatively healthy Zbtb20-null mice exhibited hypotension without obvious change of heart rate or other evidence for heart failure. Moreover, Zbtb20 deletion led to a marked reduction in heart size, left ventricular wall thickness, and cell size of cardiomyocytes, which was largely proportional to the decreased body growth. Notably, echocardiographic and hemodynamic analyses showed that cardiac contractility was greatly impaired in the absence of ZBTB20. Mechanistically, ZBTB20 deficiency decreased cardiac ATP contents, and compromised the enzyme activity of mitochondrial complex I in heart as well as L-type calcium current density in cardiomyocytes. Furthermore, the developmental activation of some mitochondrial function-related genes was significantly attenuated in Zbtb20-null myocardium, which included Hspb8, Ckmt2, Cox7a1, Tfrc, and Ogdhl. Put together, these results suggest that ZBTB20 plays a crucial role in the regulation of heart development, energy metabolism, and contractility.
    Keywords:  blood pressure; cardiomyocyte; contractility; knockout; mitochondrion; transcription factor
  10. Biochim Biophys Acta Bioenerg. 2020 Aug 25. pii: S0005-2728(20)30150-X. [Epub ahead of print] 148300
      Alteration in metabolic repertoire is associated with resistance phenotype. Although a common phenotype, not much efforts have been undertaken to design effective strategies to target the metabolic drift in cancerous cells with drug resistant properties. Here, we identified that drug resistant AML cell line HL-60/MX2 did not follow classical Warburg effect, instead these cells exhibited drastically low levels of aerobic glycolysis. Biochemical analysis confirmed reduced glucose consumption and lactic acid production by resistant population with no differences in glutamine consumption. Raman spectroscopy revealed increased lipid and cytochrome content in resistant cells which were also visualized as lipid droplets by Raman mapping, electron microscopy and lipid specific staining. Gene set enrichment analysis data from sensitive and resistant cell lines revealed significant enrichment of lipid metabolic pathways in HL-60/MX2 cells. Further, HL-60/MX2 possessed higher mitochondrial activity and increased OXPHOS suggesting the role of fatty acid metabolism as energy source which was confirmed by increased rate of fatty acid oxidation. Accordingly, OXPHOS inhibitor increased sensitivity of resistant cells to chemotherapeutic drug and fatty acid oxidation inhibitor Etomoxir reduced colony formation ability of resistant cells demonstrating the requirement of fatty acid metabolism and dependency on OXPHOS by resistant leukemic cells for survival and tumorigenicity.
    Keywords:  Drug resistance; Leukemia; Lipid metabolism; Mitochondria; OXPHOS
  11. Cell Death Dis. 2020 Aug 26. 11(8): 708
      Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.
  12. Front Cell Dev Biol. 2020 ;8 660
      Defects in mitochondrial function lead to severe neuromuscular orphan pathologies known as mitochondrial disease. Among them, Leigh Syndrome is the most common pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death. Mitochondrial diseases are characterized by a marked anatomical and cellular specificity. However, the molecular determinants for this susceptibility are currently unknown, hindering the efforts to find an effective treatment. Due to the complex crosstalk between mitochondria and their supporting cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us to assess the proteome and acetylome of a subset of susceptible neurons in a well characterized model recapitulating the human disease. Our results show a marked reduction of complex I N-module subunit abundance and an increase in the levels of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins such as PKCδ, and the complement subcomponent C1Q were also increased in Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to decreased PDH activity. We provide novel insight on the pathways involved in mitochondrial disease, which could underlie potential therapeutic approaches for these pathologies.
    Keywords:  Leigh syndrome; animal models; cell type-specific; mitochondrial isolation; neuroscience; proteomics
  13. J Biol Chem. 2020 Aug 28. pii: jbc.RA120.014483. [Epub ahead of print]
      Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide (O2 .-) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of O2 .- and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary O2 .- source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, O2 .- is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side-reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.
    Keywords:  computational biology; computer modeling; enzyme kinetics; enzyme mechanism; free radicals; hydrogen peroxide; redox regulation; succinate dehydrogenase; superoxide ion; ubiquinone
  14. Biochim Biophys Acta Bioenerg. 2020 Aug 20. pii: S0005-2728(20)30140-7. [Epub ahead of print] 148290
      We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO- production during oxidative stress. We monitored generation of ONOO- from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO- production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•-. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO- to form dityrosine (diTyr) in proportion to the ONOO- present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO- production via NO• and O2•-. Changes in mitochondrial ONOO- production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO- production, PTIO decreased production of ONOO-, and TEMPOL decreased ONOO- levels by converting more O2•- to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO- into the buffer is dependent both on O2•- released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.
    Keywords:  heart; liver mitochondria; mitochondrial oxidant stress; nitric oxide; nitric oxide synthase; peroxynitrite; superoxide
  15. EMBO Mol Med. 2020 Aug 25. e12146
      Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post-mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG-driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD+ metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.
    Keywords:   POLG ; mitochondria; mitophagy; neural stem cells; reactive oxygen species
  16. Transl Med Aging. 2020 Aug 21.
      Recent advancements in genomic, transcriptomic, proteomic, and metabolomic techniques have prompted fresh inquiry in the field of aging. Here, we outline the application of these techniques in the context of the mitochondrial genome and suggest their potential for use in exploring the biological mechanisms of the aging immune system.
    Keywords:  Genomics; Immune function; Mitochondria; Proteomics; Transcriptomics
  17. Biochem Biophys Res Commun. 2020 Sep 10. pii: S0006-291X(20)31349-8. [Epub ahead of print]530(1): 29-34
      Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.
    Keywords:  Bicarbonate; Membrane potential; Mitochondria; Protonophore; Uncoupler
  18. Annu Rev Genet. 2020 Aug 28.
      Out of many intracellular bacteria, only the mitochondria and chloroplasts abandoned their independence billions of years ago and became endosymbionts within the host eukaryotic cell. Consequently, one cannot grow eukaryotic cells without their mitochondria, and the mitochondria cannot divide outside of the cell, thus reflecting interdependence. Here, we argue that such interdependence underlies the fundamental role of mitochondrial activities in the emergence of metazoans. Several lines of evidence support our hypothesis: (a) Differentiation and embryogenesis rely on mitochondrial function; (b) mitochondrial metabolites are primary precursors for epigenetic modifications (such as methyl and acetyl), which are critical for chromatin remodeling and gene expression, particularly during differentiation and embryogenesis; (c) mitonuclear coregulation adapted to accommodate both housekeeping and tissue-dependent metabolic needs. We discuss the evolution of the unique mitochondrial genetic system, mitochondrial metabolites, mitonuclear coregulation, and their critical roles in the emergence of metazoans and in human disorders. Expected final online publication date for the Annual Review of Genetics, Volume 54 is November 23, 2020. Please see for revised estimates.
  19. Redox Biol. 2020 Aug 07. pii: S2213-2317(20)30876-4. [Epub ahead of print]36 101671
      In the present study, we hypothesized that hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy plays a protective role in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Mitophagy was evaluated by measuring the changes of mitophagy flux, mitochondria DNA copy number, and the changes of mitophagy-related proteins including translocase of outer mitochondrial membrane 20 (TOMM20), cytochrome c oxidase IV (COX IV), microtubule-associated protein 1 light chain 3B (LC3B), and mitochondria adaptor nucleoporin p62 in HK2 cells, a human tubular cell line. Results show that HIF-1α knockout significantly attenuated hypoxia/reoxygenation (H/R)-induced mitophagy, aggravated H/R-induced apoptosis, and increased the production of reactive oxygen species (ROS). Similarly, H/R induced significantly increase in Bcl-2 19-kDa interacting protein 3 (BNIP3), a downstream regulator of HIF-1α. Notably, BNIP3 overexpression reversed the inhibitory effect of HIF-1α knockout on H/R-induced mitophagy, and prevented the enhancing effect of HIF-1α knockout on H/R-induced apoptosis and ROS production. For in vivo study, we established HIF-1αflox/flox; cadherin-16-cre mice in which tubular HIF-1α was specifically knockout. It was found that tubular HIF-1α knockout significantly inhibited I/R-induced mitophagy, and aggravated I/R-induced tubular apoptosis and kidney damage. In contrast, adenovirus-mediated BNIP3 overexpression significantly reversed the decreased mitophagy, and prevented enhanced kidney damage in tubular HIF-1α knockout mice with I/R injury. In summary, our study demonstrated that HIF-1α-BNIP3-mediated mitophagy in tubular cells plays a protective role through inhibition of apoptosis and ROS production in acute kidney damage.
    Keywords:  Acute kidney injury; Bcl-2 19-kDa interacting protein 3; Hypoxia-inducible factor 1α; Mitophagy; Tubular cells
  20. Cell Commun Signal. 2020 Aug 24. 18(1): 131
      BACKGROUND: Metabolic reprogramming, in which energetic metabolism changes from oxidative phosphorylation to glycolysis, is well-accepted as a hallmark of cancers including hepatocellular carcinoma (HCC). A growing body of evidence suggests the involvement of oncogenes and tumor suppressor genes in the control of metabolic reprogramming. In this study, we attempt to investigate whether loss of PTEN, a recognized tumor suppressor, drives metabolic reprogramming of HCC.METHODS: Cancerous liver tissues were surgically resected from 128 HCC patients, with 43 adjacent noncancerous liver tissues as control. Aerobic glycolysis (Warburg effect) was reflected by measurements of glucose uptake and lactate production, mitochondrial membrane potential collapse was observed by JC-1 staining, glycolytic rate and mitochondrial respiration were evaluated by determining glycolytic proton efflux rate (glycoPER) and oxygen consumption rate (OCR) in cultured human HHCC cells.
    RESULTS: Reciprocal expression of PTEN and PI3K was determined in cancer liver tissues. Overexpression of PTEN suppressed the Warburg effect, as evidenced by reductions in glucose uptake and lactate production, maintenance of mitochondrial function, and transformation of energetic metabolism from glycolysis to oxidative phosphorylation in cultured PTEN-negative HHCC cells. Importantly, 740 Y-P, a PI3K agonist that leads to activation of the PI3K pathway, partially abrogated the function of PTEN and reprogramed glucose metabolism in cultured HHCC cells.
    CONCLUSIONS: The discovery that loss of PTEN allows the tumor metabolic program has been a major advance in understanding the carcinogenesis of HCC. Video abstract Graphic abstract showing that loss of PTEN regulates the tumor metabolic program in hepatocellular carcinoma. Loss of PTEN leads to activation of the PI3K pathway enhances the Warburg effect, thereby promoting the development of hepatocellular carcinoma.
    Keywords:  Glucose uptake; Hepatocellular carcinoma; Lactate production; Metabolic reprogramming; PI3K pathway; PTEN; Warburg effect
  21. Cancer Genomics Proteomics. 2020 Sep-Oct;17(5):17(5): 469-497
      BACKGROUND/AIM: Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway.MATERIALS AND METHODS: In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays.
    RESULTS: The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPARγ coactivator 1α (PGC-1α), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes.
    CONCLUSION: These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.
    Keywords:  Cancer; GPI; LDHA; LDHB; Warburg effect; genes; metabolism
  22. Sci Adv. 2020 Jul;6(31): eaba5345
      Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.
  23. Life (Basel). 2020 Aug 26. pii: E164. [Epub ahead of print]10(9):
      Mitochondria are complex organelles that harbour their own genome. Mitochondrial DNA (mtDNA) exists in the form of a circular double-stranded DNA molecule that must be replicated, segregated and distributed around the mitochondrial network. Human cells typically possess between a few hundred and several thousand copies of the mitochondrial genome, located within the mitochondrial matrix in close association with the cristae ultrastructure. The organisation of mtDNA around the mitochondrial network requires mitochondria to be dynamic and undergo both fission and fusion events in coordination with the modulation of cristae architecture. The dysregulation of these processes has profound effects upon mtDNA replication, manifesting as a loss of mtDNA integrity and copy number, and upon the subsequent distribution of mtDNA around the mitochondrial network. Mutations within genes involved in mitochondrial dynamics or cristae modulation cause a wide range of neurological disorders frequently associated with defects in mtDNA maintenance. This review aims to provide an understanding of the biological mechanisms that link mitochondrial dynamics and mtDNA integrity, as well as examine the interplay that occurs between mtDNA, mitochondrial dynamics and cristae structure.
    Keywords:  cristae; mitochondria; mitochondrial diseas; mitochondrial fission; mitochondrial fusion; mtDNA
  24. Nat Commun. 2020 Aug 27. 11(1): 4279
      Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.
  25. J Immunol Res. 2020 ;2020 8340329
      Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/β-catenin pathway by promoting nuclear translocation of β-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.
  26. Chem Res Toxicol. 2020 Aug 26.
      The aim of this study was to investigate the detailed mechanisms of hepatotoxicity induced by cadmium telluride quantum dots (CdTe-QDs) in BALB/c mice after intravenous injection. The study investigated oxidative stress and apoptosis, and effects on mitochondria as potential mechanistic events to elucidate the observed hepatotoxicity. Oxidative stress in the liver, induced by CdTe-QD exposure, was demonstrated by depletion of total glutathione, an increase in superoxide dismutase activity, and changes in gene expression of several oxidative stress-related biomarkers. Furthermore, CdTe-QD treatment led to apoptosis in the liver via both intrinsic and extrinsic apoptotic pathways. Effects on mitochondria were evidenced by the enlargement and increase in the number of mitochondria in hepatocytes of treated mice. CdTe-QDs also caused changes in the levels and gene expression of electron transport chain enzymes, depletion of ATP, and an increase in the level of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis. The findings from this study suggest that CdTe-QDs induced hepatotoxicity might have originated from mitochondrial effects which resulted in oxidative stress and apoptosis in the liver cells. This study provides insight into the biological effects of CdT-QDs at the tissue level and the detailed mechanisms of their toxicity in animals. The study also provides important data for bridging the gap between in vitro and in vivo testing and risk assessment of these NPs.
  27. Elife. 2020 Aug 25. pii: e56664. [Epub ahead of print]9
      Respiration, an essential metabolic process, provides cells with chemical energy. In eukaryotes, respiration occurs via the mitochondrial electron transport chain (mETC) composed of several large membrane-protein complexes. Complex I (CI) is the main entry point for electrons into the mETC. For plants, limited availability of mitochondrial material has curbed detailed biochemical and structural studies of their mETC. Here, we present the cryoEM structure of the known CI assembly intermediate CI* from Vigna radiata at 3.9 Å resolution. CI* contains CI's NADH-binding and CoQ-binding modules, the proximal-pumping module and the plant-specific γ-carbonic-anhydrase domain (γCA). Our structure reveals significant differences in core and accessory subunits of the plant complex compared to yeast, mammals and bacteria, as well as the details of the γCA domain subunit composition and membrane anchoring. The structure sheds light on differences in CI assembly across lineages and suggests potential physiological roles for CI* beyond assembly.
    Keywords:  Vigna radiata; assembly; biochemistry; chemical biology; complex I; electron microscopy; mitochondria; molecular biophysics; respiration; structural biology
  28. Toxicol Lett. 2020 Aug 21. pii: S0378-4274(20)30397-0. [Epub ahead of print]
      Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, hepatotoxic and immunotoxic effects. We previously demonstrated that OTA could cause mitochondrial function disturbance in GES-1 cells in vitro, which lead to the presumption that the glucose metabolism of GES-1 cells will be altered by OTA. Therefore in the present study, we explored the toxicity of OTA on glucose metabolism of GES-1 cells and the molecular mechanism. We found that OTA could induce aerobic glycolysis, evidenced shown by increase of glucose consumption, lactate production and cellular ATP concentration. We further detected expressions of GLUT1 and glycolytic enzymes including HK2, PFK1, PKM2 and LDHA as well as tricarboxylic acid (TCA) cycle-associated enzymes including IDH1, OGDH and CS. The results showed that expression of GLUT1 as well as the activities and expressions of HK2, PFK1 and LDHA were significantly increased while IDH1 and OGDH were reduced by OTA. As to PKM2, western blot showed that OTA could elevated the phospho-PKM2 Ser37 protein level and induce the nuclear accumulation of PKM2, which was further supported by immunofluorescence analyses, in addition, pyruvate kinase activity was reduced by OTA. In conclusion, these findings suggest that OTA exposure induces the metabolic shift from oxidative phosphorylation to aerobic glycolysis via regulating the activities and expressions of glycolysis and TCA-cycle associated molecules in GES-1 cells.
    Keywords:  Aerobic glycolysis; Glucose metabolism; Ochratoxin A; Tricarboxylic acid cycle
  29. Aging (Albany NY). 2020 Aug 27. 12
      It is estimated that over 44 million people across the globe have dementia, and half of these cases are believed to be Alzheimer's disease (AD). As the proportion of the global population which is over the age 60 increases so will the number of individuals living with AD. This will result in ever-increasing demands on healthcare systems and the economy. AD can be either sporadic or familial, but both present with similar pathobiology and symptoms. Three prominent theories about the cause of AD are the amyloid, tau and mitochondrial hypotheses. The mitochondrial hypothesis focuses on mitochondrial dysfunction in AD, however little attention has been given to the potential dysfunction of the mitochondrial ATP synthase in AD. ATP synthase is a proton pump which harnesses the chemical potential energy of the proton gradient across the inner mitochondrial membrane (IMM), generated by the electron transport chain (ETC), in order to produce the cellular energy currency ATP. This review presents the evidence accumulated so far that demonstrates dysfunction of ATP synthase in AD, before highlighting two potential pharmacological interventions which may modulate ATP synthase.
    Keywords:  ATP synthase; Alzheimer's disease; mitochondria
  30. EMBO Rep. 2020 Aug 27. e48483
      MICU1 is a mitochondrial inner membrane protein that inhibits mitochondrial calcium entry; elevated MICU1 expression is characteristic of many cancers, including ovarian cancer. MICU1 induces both glycolysis and chemoresistance and is associated with poor clinical outcomes. However, there are currently no available interventions to normalize aberrant MICU1 expression. Here, we demonstrate that microRNA-195-5p (miR-195) directly targets the 3' UTR of the MICU1 mRNA and represses MICU1 expression. Additionally, miR-195 is under-expressed in ovarian cancer cell lines, and restoring miR-195 expression reestablishes native MICU1 levels and the associated phenotypes. Stable expression of miR-195 in a human xenograft model of ovarian cancer significantly reduces tumor growth, increases tumor doubling times, and enhances overall survival. In conclusion, miR-195 controls MICU1 levels in ovarian cancer and could be exploited to normalize aberrant MICU1 expression, thus reversing both glycolysis and chemoresistance and consequently improving patient outcomes.
    Keywords:  MICU1/CBARA1; Ovarian cancer; chemoresistance; glycolysis; miR-195
  31. Sci Rep. 2020 Aug 24. 10(1): 14092
      Colorectal cancer (CRC) is a common malignancy occurring in the digestive system. Despite progress in surgery and therapy options, CRC is still a considerable cause of cancer mortality worldwide. In this study, a colon cancer patient-derived xenograft model was established to evaluate the antitumor activity of Shikonin. The protective effect underlying Shikonin was determined through assessing serum levels of liver enzymes (ALT, AST) and kidney functions (BuN, Scr) in PDX mice. Proteomics and metabolomics profiles were integrated to provide a systematic perspective in dynamic changes of proteins and global endogenous metabolites as well as their perturbed pathways. A total of 456 differently expressed proteins (DEPs), 32 differently expressed metabolites (DEMs) in tumor tissue, and 20 DEMs in mice serum were identified. The perturbation of arginine biosynthesis, purine metabolism, and biosynthesis of amino acids may mainly account for therapeutic mechanism of Shikonin. Furthermore, the expression of mRNAs participating in arginine biosynthesis (CPS1, OTC, Arg1) and do novo purine synthesis (GART, PAICS, ATIC) were validated through RT-qPCR. Our study provides new insights into the drug therapeutic strategies and a better understanding of antitumor mechanisms that might be valuable for further studies on Shikonin in the clinical treatment of colorectal cancer.
  32. Nat Commun. 2020 Aug 27. 11(1): 4281
      Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.
  33. Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31058-5. [Epub ahead of print]32(8): 108073
      Immune checkpoint blockade (ICB) has led to therapeutic responses in some cancer patients for whom no effective treatment previously existed. ICB acts on T lymphocytes and other immune cells that are inactivated due to checkpoint signals that inhibit their infiltration and function within tumors. But for more than 80% of patients, immunotherapy has not been effective. Here, we demonstrate a cancer-cell-intrinsic mechanism of immune evasion and resistance to ICB mediated by baculoviral IAP repeat-containing 2 (BIRC2). Knockdown of BIRC2 expression in mouse melanoma or breast cancer cells increases expression of the chemokine CXCL9 and impairs tumor growth by increasing the number of intratumoral activated CD8+ T cells and natural killer cells. Administration of anti-CXCL9 neutralizing antibody inhibits the recruitment of CD8+ T cells and natural killer cells to BIRC2-deficient tumors. Most importantly, BIRC2 deficiency dramatically increases the sensitivity of mouse melanoma and breast tumors to anti-CTLA4 and/or anti-PD1 ICB.
    Keywords:  NF-κB; anti-tumor immunity; cancer immunosuppression; hypoxia-inducible factors; tumor microenvironment
  34. J Biol Chem. 2020 Aug 23. pii: jbc.RA120.012618. [Epub ahead of print]
      Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N-terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast differentiation, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates nicotinamide adenine dinucleotide (NAD) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells.
    Keywords:  NOCT; Nocturnin; gene regulation; mRNA decay; mitochondria; nicotinamide adenine dinucleotide (NAD); ribonuclease
  35. Front Physiol. 2020 ;11 950
      The accumulation of lipid droplets in the cytoplasm of hepatocytes, known as hepatic steatosis, is a hallmark of non-alcoholic fatty liver disease (NAFLD). Inhibiting hepatic steatosis is suggested to be a therapeutic strategy for NAFLD. The present study investigated the actions of Neurotropin (NTP), a drug used for chronic pain in Japan and China, on lipid accumulation in hepatocytes as a possible treatment for NAFLD. NTP inhibited lipid accumulation induced by palmitate and linoleate, the two major hepatotoxic free fatty acids found in NAFLD livers. An RNA sequencing analysis revealed that NTP altered the expression of mitochondrial genes. NTP ameliorated palmitate-and linoleate-induced mitochondrial dysfunction by reversing mitochondrial membrane potential, respiration, and β-oxidation, suppressing mitochondrial oxidative stress, and enhancing mitochondrial turnover. Moreover, NTP increased the phosphorylation of AMPK, a critical factor in the regulation of mitochondrial function, and induced PGC-1β expression. Inhibition of AMPK activity and PGC-1β expression diminished the anti-steatotic effect of NTP in hepatocytes. JNK inhibition could also be associated with NTP-mediated inhibition of lipid accumulation, but we did not find the association between AMPK and JNK. These results suggest that NTP inhibits lipid accumulation by maintaining mitochondrial function in hepatocytes via AMPK activation, or by inhibiting JNK.
    Keywords:  AMPK; fatty liver; lipid metabolism; liver; mitochondria
  36. Biochem Biophys Res Commun. 2020 Sep 10. pii: S0006-291X(20)31471-6. [Epub ahead of print]530(1): 285-291
      Recent advances in cancer biology reveal the importance of metabolic changes in cancer; however, less is known about how metabolic pathways in tumors are regulated in vivo. Here, we report analysis of the lung cancer metabolism based on different surgical procedures, namely lobectomy and partial resection. In lobectomy, but not in partial resection, pulmonary arteries and veins are ligated prior to removal of tissues, rendering tissues ischemic. We show that tumors indeed undergo ischemia upon lobectomy and that the tumor metabolome differs markedly from that of tumors removed by partial resection. Comparison of the responses to ischemia in tumor and normal lung tissues revealed that lung cancer tissue exhibits greater TCA cycle and autophagic activity than do normal lung tissues in vivo in patients. Finally, we report that deleting ATG7, which encodes a protein essential for autophagy, antagonizes growth of tumors derived from lung cancer cell lines, suggesting that autophagy confers metabolic advantages to lung cancer. Our findings shed light on divergent metabolic responses to ischemia seen in tumors and normal tissues.
    Keywords:  Autophagy; Cancer metabolism; Ischemia; Lung cancer; Metabolome
  37. PLoS One. 2020 ;15(8): e0235551
      VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.
  38. Methods Mol Biol. 2021 ;2202 33-42
      Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals.
    Keywords:  Lipid peroxidation; Live cell imaging; Mitochondria; ROS production
  39. Curr Pharm Des. 2020 Aug 26.
      Mitochondria are maternally inherited semi-autonomous organelles that play a central role in redox balance, energy metabolism, control of integrated stress responses, and cellular homeostasis. The molecular communication between mitochondria and the nucleus is intricate and bidirectional in nature. Though mitochondrial genome encodes for a number of key proteins involved in oxidative phosphorylation, several regulatory factors encoded by nuclear DNA are prominent contributors to mitochondrial biogenesis and function. The loss of synergy between this reciprocal control of anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signaling, triggers epigenomic imbalance, and affects mitochondrial function and global gene expressions. Recent expansions of our knowledge on mitochondrial epigenomics have offered novel perspectives for the study of several non-communicable diseases including cancer. As mitochondria are considered beacons for pharmacological interventions, new frontiers in targeted delivery approaches could provide opportunities for effective disease management and cure through reversible epigenetic reprogramming. This review focuses on recent progress in the area of mitochondrial-nuclear cross-talk and epigenetic regulation of mitochondrial DNA methylation, mitochondrial micro RNAs, and post-translational modification of mitochondrial nucleoid-associated proteins that hold major opportunities for targeted drug delivery and clinical translation.
    Keywords:  Epigenomicsignatures; Mitochondrial medicine; Mitochondrial targeting; Mitochondrial-nuclear cross-talk; Translational research
  40. FASEB J. 2020 Aug 29.
      Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so-called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)-adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET-formation. Moreover, we characterized the mechanism leading to alum-induced NET-release in human neutrophils as rapid, NADPH oxidase-independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+ -flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET-release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum-containing vaccines in humans and provides novel insights into bioenergetic requirements of NET-formation.
    Keywords:  NET; adjuvant; aluminium hydroxide; innate response; neutrophils; vaccine
  41. Cancer Res. 2020 Aug 28. pii: canres.3971.2019. [Epub ahead of print]
      Amplification and expression of the MYC oncogene in tumor cells, including ovarian cancer cells, correlates with poor responses to chemotherapy. As MYC is not directly targetable, we have analyzed molecular pathways downstream of MYC to identify potential therapeutic targets. Here we report that ovarian cancer cells overexpressing glutaminase (GLS), a MYC target and a key enzyme in glutaminolysis, are intrinsically resistant to platinum chemotherapy and are enriched in the intracellular antioxidant glutathione. Deprivation of glutamine by glutamine-withdrawal, GLS knockdown, or exposure to the GLS inhibitor CB-839 resulted in robust induction of reactive oxygen species in high GLS-expressing but not low GLS-expressing ovarian cancer cells. Treatment with CB-839 rendered GLShigh cells vulnerable to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib and prolonged survival in tumor-bearing mice. These findings suggest that applying a combined therapy of GLS inhibitor and PARP inhibitor could effectively treat chemoresistant ovarian cancers, especially those with high expression of GLS.
  42. Am J Physiol Renal Physiol. 2020 Aug 24.
      Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis, and the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI specific siRNA in the absence and presence of an antioxidant tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in the PDI inhibited cells. Also, PDI inhibition caused Nrf2 (nuclear factor E2 related factor 2, a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase, and glutathione S-transferase activities, and increased oxidative stress. In PDI inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress, and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, while Keap1 silencing (Kelch-like ECH-associated protein 1, a Nrf2 regulatory protein) protected the cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. These studies suggest an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.
    Keywords:  Apoptosis; Keap1/Nrf2; Mitochondria; Oxidative Stress; PDI
  43. Nat Commun. 2020 Aug 28. 11(1): 4337
      Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure.
  44. Nat Commun. 2020 Aug 27. 11(1): 4306
      Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy.
  45. Proc Natl Acad Sci U S A. 2020 Aug 24. pii: 202003537. [Epub ahead of print]
      Hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (MRSI) is a noninvasive metabolic-imaging modality that probes carbon flux in tissues and infers the state of metabolic reprograming in tumors. Prevailing models attribute elevated hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in aggressive tumors to enhanced glycolytic flux and lactate dehydrogenase A (LDHA) activity (Warburg effect). By contrast, we find by cross-sectional analysis using genetic and pharmacological tools in mechanistic studies applied to well-defined genetically engineered cell lines and tumors that initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates as well as global conversion were highly dependent on and critically rate-limited by the transmembrane influx of [1-13C]pyruvate mediated predominately by monocarboxylate transporter-1 (MCT1). Specifically, in a cell-encapsulated alginate bead model, induced short hairpin (shRNA) knockdown or overexpression of MCT1 quantitatively inhibited or enhanced, respectively, unidirectional pyruvate influxes and [1-13C]pyruvate-to-[1-13C]lactate conversion rates, independent of glycolysis or LDHA activity. Similarly, in tumor models in vivo, hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion was highly dependent on and critically rate-limited by the induced transmembrane influx of [1-13C]pyruvate mediated by MCT1. Thus, hyperpolarized [1-13C]pyruvate MRSI measures primarily MCT1-mediated [1-13C]pyruvate transmembrane influx in vivo, not glycolytic flux or LDHA activity, driving a reinterpretation of this maturing new technology during clinical translation. Indeed, Kaplan-Meier survival analysis for patients with pancreatic, renal, lung, and cervical cancers showed that high-level expression of MCT1 correlated with poor overall survival, and only in selected tumors, coincident with LDHA expression. Thus, hyperpolarized [1-13C]pyruvate MRSI provides a noninvasive functional assessment primarily of MCT1 as a clinical biomarker in relevant patient populations.
    Keywords:  MCT1; [1-13C]pyruvate; hyperpolarized NMR; imaging biomarker; monocarboxylate transporters
  46. iScience. 2020 Aug 13. pii: S2589-0042(20)30646-5. [Epub ahead of print]23(9): 101454
      During an immune response, natural killer (NK) cells activate specific metabolic pathways to meet the increased energetic and biosynthetic demands associated with effector functions. Here, we found in vivo activation of NK cells during Listeria monocytogenes infection-augmented transcription of genes encoding mitochondria-associated proteins in a manner dependent on the transcriptional coactivator PGC-1α. Using an Ncr1Cre-based conditional knockout mouse, we found that PGC-1α was crucial for optimal NK cell effector functions and bioenergetics, as the deletion of PGC-1α was associated with decreased cytotoxic potential and cytokine production along with altered ADP/ATP ratios. Lack of PGC-1α also significantly impaired the ability of NK cells to control B16F10 tumor growth in vivo, and subsequent gene expression analysis showed that PGC-1α mediates transcription required to maintain mitochondrial activity within the tumor microenvironment. Together, these data suggest that PGC-1α-dependent transcription of specific target genes is required for optimal NK cell function during the response to infection or tumor growth.
    Keywords:  Biological Sciences; Cancer; Cellular Physiology; Immunology
  47. Nat Cancer. 2020 Mar;1(3): 345-358
      Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal organoids to promote stemness and leads to adenocarcinoma formation in vivo via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation. Mechanistically, we found that aKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using CRC patient-derived organoids and several in vivo CRC tumour models, we show that aKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumour growth and extending survival. Together, our results reveal how metabolic microenvironment impacts Wnt signaling and identify aKG as a potent antineoplastic metabolite for potential differentiation therapy for CRC patients.
    Keywords:  Wnt signaling; cancer metabolism; colon cancer; epigenetics; glutamine
  48. EMBO Rep. 2020 Aug 27. e50964
      Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?
  49. Nat Commun. 2020 Aug 27. 11(1): 4289
      Older organs represent an untapped potential to close the gap between demand and supply in organ transplantation but are associated with age-specific responses to injury and increased immunogenicity, thereby aggravating transplant outcomes. Here we show that cell-free mitochondrial DNA (cf-mt-DNA) released by senescent cells accumulates with aging and augments immunogenicity. Ischemia reperfusion injury induces a systemic increase of cf-mt-DNA that promotes dendritic cell-mediated, age-specific inflammatory responses. Comparable events are observed clinically, with the levels of cf-mt-DNA elevated in older deceased organ donors, and with the isolated cf-mt-DNA capable of activating human dendritic cells. In experimental models, treatment of old donor animals with senolytics clear senescent cells and diminish cf-mt-DNA release, thereby dampening age-specific immune responses and prolonging the survival of old cardiac allografts comparable to young donor organs. Collectively, we identify accumulating cf-mt-DNA as a key factor in inflamm-aging and present senolytics as a potential approach to improve transplant outcomes and availability.
  50. Metabolomics. 2020 Aug 26. 16(9): 91
      INTRODUCTION: Repurposing of cationic amphiphilic drugs (CADs) emerges as an attractive therapeutic solution against various cancers, including leukemia. CADs target lysosomal lipid metabolism and preferentially kill cancer cells via induction of lysosomal membrane permeabilization, but the exact effects of CADs on the lysosomal lipid metabolism remain poorly illuminated.OBJECTIVES: We aimed to systematically monitor CAD-induced alterations in the quantitative lipid profiles of leukemia cell lines in order to chart effects of CADs on the metabolism of various lipid classes present in these cells.
    METHODS: We conducted this study on eight cultured cell lines representing two leukemia types, acute lymphoblastic leukemia and acute myeloid leukemia. Mass spectrometry-based quantitative shotgun lipidomics was employed to quantify the levels of around 400 lipid species of 26 lipid classes in the leukemia cell lines treated or untreated with a CAD, siramesine.
    RESULTS: The two leukemia types displayed high, but variable sensitivities to CADs and distinct profiles of cellular lipids. Treatment with siramesine rapidly altered the levels of diverse lipid classes in both leukemia types. These included sphingolipid classes previously reported to play key roles in CAD-induced cell death, but also lipids of other categories. We demonstrated that the treatment with siramesine additionally elevated the levels of numerous cytolytic lysoglycerophospholipids in positive correlation with the sensitivity of individual leukemia cell lines to siramesine.
    CONCLUSIONS: Our study shows that CAD treatment alters balance in the metabolism of glycerophospholipids, and proposes elevation in the levels of lysoglycerophospholipids as part of the mechanism leading to CAD-induced cell death of leukemia cells.
    Keywords:  Cancer; Lipidomics; Lysoglycerophospholipids; Lysosomes; Sphingolipids; Systems biology
  51. Nat Commun. 2020 Aug 28. 11(1): 4319
      Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.
  52. Biochim Biophys Acta Mol Basis Dis. 2020 Aug 19. pii: S0925-4439(20)30283-0. [Epub ahead of print] 165935
      Deregulation of nutrient, hormonal, or neuronal signaling produces metabolic alterations that result in increased mitochondrial reactive oxygen species (ROS) production. The associations of the mitochondrial respiratory chain components into supercomplexes could have pathophysiological relevance in metabolic diseases, as supramolecular arrangements, by sustaining a high electron transport rate, might prevent ROS generation. In this review, the relationship between mitochondrial dysfunction and supercomplex arrangement of the mitochondrial respiratory chain components in obesity, insulin resistance, hepatic steatosis and diabetes mellitus is summarized and discussed.
    Keywords:  Insulin resistance; Liver; Obesity; Respiratory supercomplexes; T2DM
  53. Nat Commun. 2020 Aug 27. 11(1): 4296
      Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.
  54. Anal Chem. 2020 Aug 23.
      Herein we describe the development of the first mitochondrial Cl--selective fluorescent probe, Mito-MQAE, and its applications in biological systems. Fluorescence of Mito-MQAE is insensitive to pH over the physiological pH range and is quenched by Cl- with a Stern-Volmer quenching constant of 201 M-1 at pH 7.0. The results of cell studies using Mito-MQAE show that substances with the ability to disrupt mitochondrial membranes cause increases in the mitochondrial Cl- concentration.
  55. Neoplasma. 2020 Aug 28. pii: 200302N216. [Epub ahead of print]
      There is a great effort to connect the accumulation of 2-hydroxyglutarate (2-HG) oncometabolite with cellular onco-epigenetic status and subsequently predict the prognoses of glioma patients. In this observational study, the concentrations of D- and L- 2-HG were determined in 57 tumor tissue samples of glioma patients (n = 57) WHO grade I through IV (astrocytoma, oligodendroglioma, secondary glioblastoma and glioblastoma multiforme) in vitro. Also, genetic mutation status on isocitrate dehydrogenase 1 and 2 (IDH 1/2) was determined from these samples. The objective of this study was to confirm or to reject the hypothesis of the direct correlation of 2-HG concentration in tumor tissue and the results from IDH 1/IDH 2 point mutation analyses. The concentrations of 2-HG were quantified using high sensitive HPLC and Q-TOF HRMS spectrometer setup. Concurrently, the genetic mutation analyses of both IDH 1 (cytosolic) and IDH 2 (mitochondrial) were performed by the isolation of tumor tissue DNA, PCR amplification, and subsequent Sanger forward sequencing. Our results indicate that there is no definite correlation between the two as we identified cases of glioma tumors with significantly increased concentration of one or both L- and D- 2-HG but no IDH 1/2 mutations (44% 2-HG positive cases).
  56. JCI Insight. 2020 Aug 25. pii: 139628. [Epub ahead of print]
      Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hindlimb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared to the BL6 parental strain. AAV-mediated over-expression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the Glucose Metabolism Reactome. Muscles from these patients express lower PFKFB3 protein and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow related mitochondrial function is compromised pre-clinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of CLI patient limb skeletal muscle.
    Keywords:  Atherosclerosis; Cardiovascular disease; Glucose metabolism; Muscle Biology; Vascular Biology