bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2019‒09‒29
thirty-five papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Nat Commun. 2019 Sep 25. 10(1): 4346
    Li H, Bullock K, Gurjao C, Braun D, Shukla SA, Bossé D, Lalani AA, Gopal S, Jin C, Horak C, Wind-Rotolo M, Signoretti S, McDermott DF, Freeman GJ, Van Allen EM, Schreiber SL, Stephen Hodi F, Sellers WR, Garraway LA, Clish CB, Choueiri TK, Giannakis M.
      Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase   (IDO/TDO) inhibitors.
  2. Nat Commun. 2019 Sep 25. 10(1): 4341
    Urbani A, Giorgio V, Carrer A, Franchin C, Arrigoni G, Jiko C, Abe K, Maeda S, Shinzawa-Itoh K, Bogers JFM, McMillan DGG, Gerle C, Szabò I, Bernardi P.
      The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.
  3. Cancer Res. 2019 Sep 27. pii: canres.0644.2019. [Epub ahead of print]
    McGregor GH, Campbell AD, Fey SK, Tumanov S, Sumpton D, Rodriguez Blanco G, Mackay G, Nixon C, Vazquez A, Sansom OJ, Kamphorst JJ.
      Statins are widely prescribed inhibitors of the mevalonate pathway, acting to lower systemic cholesterol levels. The mevalonate pathway is critical for tumorigenesis and is frequently upregulated in cancer. Nonetheless, reported effects of statins on tumor progression are ambiguous, making it unclear if statins, alone or in combination, can be used for chemotherapy. Here, using advanced mass spectrometry and isotope tracing, we showed that statins only modestly affected cancer cholesterol homeostasis. Instead, they significantly reduced synthesis and levels of another downstream product, the mitochondrial electron carrier coenzyme Q, both in cultured cancer cells and tumors. This compromised oxidative phosphorylation, causing severe oxidative stress. To compensate, cancer cells upregulated antioxidant metabolic pathways, including reductive carboxylation, proline synthesis, and cystine import. Targeting cystine import with an xCT transporter-lowering MEK inhibitor, in combination with statins, caused profound tumor cell death. Thus, statin-induced ROS production in cancer cells can be exploited in a combinatorial regimen.
  4. Nat Chem Biol. 2019 Oct;15(10): 1001-1008
    Park JO, Tanner LB, Wei MH, Khana DB, Jacobson TB, Zhang Z, Rubin SA, Li SH, Higgins MB, Stevenson DM, Amador-Noguez D, Rabinowitz JD.
      Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines 2H and 13C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.
  5. Front Endocrinol (Lausanne). 2019 ;10 570
    Dai W, Jiang L.
      Metabolism describes the life-sustaining chemical reactions in organisms that provide both energy and building blocks for cellular survival and proliferation. Dysregulated metabolism leads to many life-threatening diseases including obesity, diabetes, and cancer. Mitochondria, subcellular organelles, contain the central energy-producing metabolic pathway, the tricarboxylic acid (TCA) cycle. Also, mitochondria exist in a dynamic network orchestrated by extracellular nutrient levels and intracellular energy needs. Upon stimulation, mitochondria undergo consistent interchange through fusion (small to big) and fission (big to small) processes. Mitochondrial fusion is primarily controlled by three GTPases, mitofusin 1 (Mfn1), Mfn2, and optic atrophy 1 (Opa1), while mitochondrial fission is primarily regulated by GTPase dynamin-related protein 1 (Drp1). Dysregulated activity of these GTPases results in disrupted mitochondrial dynamics and cellular metabolism. This review will update the metabolic roles of these GTPases in obesity, diabetes, and cancer.
    Keywords:  GTPase; TCA cycle; energy metabolism; mdivi-1; mitochondrial dynamics
  6. Expert Rev Mol Med. 2019 Sep 27. 21 e4
    Unterlass JE, Curtin NJ.
      Warburg and coworkers' observation of altered glucose metabolism in tumours has been neglected for several decades, which, in part, was because of an initial misinterpretation of the basis of their finding. Following the realisation that genetic alterations are often linked to metabolism, and that the tumour micro-environment imposes different demands on cancer cells, has led to a reinvestigation of cancer metabolism in recent years. Increasing our understanding of the drivers and consequences of the Warburg effect in cancer and beyond will help to identify new therapeutic strategies as well as to identify new prognostic and therapeutic biomarkers. Here we discuss the initial findings of Warburg and coworkers regarding cancer cell glucose metabolism, how these studies came into focus again in recent years following the discovery of metabolic oncogenes, and the therapeutic potential that lies within targeting the altered metabolic phenotype in cancer. In addition, another essential nutrient in cancer metabolism, glutamine, will be discussed.
    Keywords:  Cancer metabolism; Warburg effect; glucose metabolism; glutamine metabolism; isocitrate dehydrogenase; phosphoglycerate dehydrogenase
  7. F1000Res. 2019 ;pii: F1000 Faculty Rev-1621. [Epub ahead of print]8
    Ji LL, Yeo D.
      It is well established that mitochondria play a critical role in the metabolic and physiological adaptation of skeletal muscle to enhanced contractile activity. Several redox-sensitive signaling pathways such as PGC-1α, AMPK, IGF/Akt/mTOR, SIRT, NFκB, and FoxO are involved with extensive crosstalk to regulate vital cellular functions such as mitochondrial biogenesis, mitochondrial fusion and fission dynamics, autophagy/mitophagy, and apoptosis under altered demand and stress. However, when muscles cease contraction, such as during immobilization and denervation, mitochondria undergo a series of detrimental changes characterized by downregulation of PGC-1α and antioxidant defense, increased ROS generation, activated FoxO, NFκB, and inflammation, enhanced ubiquitination, and finally mitophagy and apoptotic cascades. The phenotypic outcome of the discord of mitochondrial homeostasis is elevated proteolysis and muscle atrophy. The demonstration that PGC-1α overexpression via transgene or in vivo DNA transfection can restore mitochondrial homeostasis and reverse myocyte atrophy supports the "mitostasis theory of muscle atrophy".
    Keywords:  Atrophy; Mitochondria; Muscle; PGC-1α; Redox Signaling
  8. EBioMedicine. 2019 Sep 14. pii: S2352-3964(19)30615-2. [Epub ahead of print]
    Seo JH, Agarwal E, Chae YC, Lee YG, Garlick DS, Storaci AM, Ferrero S, Gaudioso G, Gianelli U, Vaira V, Altieri DC.
      BACKGROUND: Mitochondrial functions are exploited in cancer and provide a validated therapeutic target. However, how this process is regulated has remained mostly elusive and the identification of new pathways that control mitochondrial integrity in cancer is an urgent priority.METHODS: We studied clinically-annotated patient series of primary and metastatic prostate cancer, representative cases of multiple myeloma (MM) and publicly available genetic databases. Gene regulation studies involved chromatin immunoprecipitation, PCR amplification and Western blotting of conditional Myc-expressing cell lines. Transient or stable gene silencing was used to quantify mitochondrial functions in bioenergetics, outer membrane permeability, Ca2+ homeostasis, redox balance and cell death. Tumorigenicity was assessed by cell proliferation, colony formation and xenograft tumour growth.
    FINDINGS: We identified Mitochondrial Fission Factor (MFF) as a novel transcriptional target of oncogenic Myc overexpressed in primary and metastatic cancer, compared to normal tissues. Biochemically, MFF isoforms, MFF1 and MFF2 associate with the Voltage-Dependent Anion Channel-1 (VDAC1) at the mitochondrial outer membrane, in vivo. Disruption of this complex by MFF silencing induces general collapse of mitochondrial functions with increased outer membrane permeability, loss of inner membrane potential, Ca2+ unbalance, bioenergetics defects and activation of cell death pathways. In turn, this inhibits tumour cell proliferation, suppresses colony formation and reduces xenograft tumour growth in mice.
    INTERPRETATION: An MFF-VDAC1 complex is a novel regulator of mitochondrial integrity and actionable therapeutic target in cancer.
    Keywords:  Cancer therapy; Cell death; MFF; Mitochondria; Tumour metabolism; VDAC1
  9. BJR Case Rep. 2019 Sep;5(3): 20190026
    Abeyakoon O, Latifoltojar A, Gong F, Papoutsaki MV, Chowdhury R, Glaser M, Jeraj H, Awais R, Holt C, Twyman F, Arstad E, Gadian DG, Atkinson D, Comment A, O'Callaghan J, Smith L, Beeston T, Clemente J, Patani N, Stein R, Yuneva M, Szabadkai G, Halligan S, Punwani S.
      Hyperpolarised 13C MRI (HP-MRI) is a novel imaging technique that allows real-time analysis of metabolic pathways in vivo.1 The technology to conduct HP-MRI in humans has recently become available and is starting to be clinically applied. As knowledge of molecular biology advances, it is increasingly apparent that cancer cell metabolism is related to disease outcomes, with lactate attracting specific attention. 2 Recent reviews of breast cancer screening programs have raised concerns and increased public awareness of over treatment. The scientific community needs to shift focus from improving cancer detection alone to pursuing novel methods of distinguishing aggressive breast cancers from those which will remain indolent. HP-MRI offers the opportunity to identify aggressive tumour phenotypes and help monitor/predict therapeutic response. Here we report one of the first cases of breast cancer imaged using HP-MRI alongside correlative conventional imaging, including breast MRI.
  10. Front Oncol. 2019 ;9 848
    Pupo E, Avanzato D, Middonti E, Bussolino F, Lanzetti L.
      Tumors driven by mutant KRAS are among the most aggressive and refractory to treatment. Unfortunately, despite the efforts, targeting alterations of this GTPase, either directly or by acting on the downstream signaling cascades, has been, so far, largely unsuccessful. However, recently, novel therapeutic opportunities are emerging based on the effect that this oncogenic lesion exerts in rewiring the cancer cell metabolism. Cancer cells that become dependent on KRAS-driven metabolic adaptations are sensitive to the inhibition of these metabolic routes, revealing novel therapeutic windows of intervention. In general, mutant KRAS fosters tumor growth by shifting cancer cell metabolism toward anabolic pathways. Depending on the tumor, KRAS-driven metabolic rewiring occurs by up-regulating rate-limiting enzymes involved in amino acid, fatty acid, or nucleotide biosynthesis, and by stimulating scavenging pathways such as macropinocytosis and autophagy, which, in turn, provide building blocks to the anabolic routes, also maintaining the energy levels and the cell redox potential (1). This review will discuss the most recent findings on mutant KRAS metabolic reliance in tumor models of pancreatic and non-small-cell lung cancer, also highlighting the role that these metabolic adaptations play in resistance to target therapy. The effects of constitutive KRAS activation in glycolysis elevation, amino acids metabolism reprogramming, fatty acid turnover, and nucleotide biosynthesis will be discussed also in the context of different genetic landscapes.
    Keywords:  KRAS; NSCLC; PDAC; gluocose metabolism in cancer; glycolysis; metabolic adaptability in cancer; metabolic rewiring
  11. Cells. 2019 Sep 12. pii: E1072. [Epub ahead of print]8(9):
    Calì T, Ottolini D, Vicario M, Catoni C, Vallese F, Cieri D, Barazzuol L, Brini M.
      Familial Parkinson's disease (PD) is associated with duplication or mutations of α-synuclein gene, whose product is a presynaptic cytosolic protein also found in mitochondria and in mitochondrial-associated ER membranes. We have originally shown the role of α-syn as a modulator of the ER-mitochondria interface and mitochondrial Ca2+ transients, suggesting that, at mild levels of expression, α-syn sustains cell metabolism. Here, we investigated the possibility that α-syn action on ER-mitochondria tethering could be compromised by the presence of PD-related mutations. The clarification of this aspect could contribute to elucidate key mechanisms underlying PD. The findings reported so far are not consistent, possibly because of the different methods used to evaluate ER-mitochondria connectivity. Here, the effects of the PD-related α-syn mutations A53T and A30P on ER-mitochondria relationship were investigated in respect to Ca2+ handling and mitochondrial function using a newly generated SPLICS sensor and aequorin-based Ca2+measurements. We provided evidence that A53T and A30P amino acid substitution does not affect the ability of α-syn to enhance ER/mitochondria tethering and mitochondrial Ca2+ transients, but that this action was lost as soon as a high amount of TAT-delivered A53T and A30P α-syn mutants caused the redistribution of α-syn from cytoplasm to foci. Our results suggest a loss of function mechanism and highlight a possible connection between α-syn and ER-mitochondria Ca2+ cross-talk impairment to the pathogenesis of PD.
    Keywords:  ER-mitochondria contact sites; Parkinson’s disease; alpha-synuclein; calcium; mitochondria
  12. Nat Commun. 2019 Sep 25. 10(1): 4363
    Li N, Wang Y, Neri S, Zhen Y, Fong LWR, Qiao Y, Li X, Chen Z, Stephan C, Deng W, Ye R, Jiang W, Zhang S, Yu Y, Hung MC, Chen J, Lin SH.
      The LKB1/AMPK pathway plays a major role in cellular homeostasis and tumor suppression. Down-regulation of LKB1/AMPK occurs in several human cancers and has been implicated in metabolic diseases. However, the precise upstream regulation of LKB1-AMPK pathway is largely unknown. Here, we report that AMPK activation by LKB1 is regulated by tankyrases. Tankyrases interact with and ribosylate LKB1, promoting its K63-linked ubiquitination by an E3 ligase RNF146, which blocks LKB1/STRAD/MO25 complex formation and LKB1 activation. LKB1 activation by tankyrase inhibitors induces AMPK activation and suppresses tumorigenesis. Similarly, the tankyrase inhibitor G007-LK effectively regulates liver metabolism and glycemic control in diabetic mice in a LKB1-dependent manner. In patients with lung cancer, tankyrase levels negatively correlate with p-AMPK levels and poor survival. Taken together, these findings suggest that tankyrase and RNF146 are major up-stream regulators of LKB1-AMPK pathway and provide another focus for cancer and metabolic disease therapies.
  13. Cell Metab. 2019 Sep 17. pii: S1550-4131(19)30495-4. [Epub ahead of print]
    Chen S, Henderson A, Petriello M, Romano KA, Gearing M, Miao J, Schell M, Sandoval-EspinolaEspinola WJ, Tao J, Sha B, Graham M, Crooke R, Kleinridders A, Balskus EP, Rey FE, Morris A, Biddinger SB.
      The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.
    Keywords:  EIF2AK3; FoxO1; PERK; diabetes; endoplasmic reticulum stress; insulin signaling; metabolomics; trimethylamine N-oxide
  14. Mol Cancer Ther. 2019 Sep 23. pii: molcanther.0103.2019. [Epub ahead of print]
    Tang X, Fu X, Liu Y, Yu D, Cai SJ, Yang C.
      Mutations in genes encoding isocitrate dehydrogenases (IDHs) 1 and 2 are common cancer-related genetic abnormalities. Malignancies with mutated IDHs exhibit similar pathogenesis, metabolic pattern, and resistance signature. However, an effective therapy against IDH1-mutated solid tumor remains unavailable. In the present study, we showed that acquisition of IDH1 mutation results in the disruption of NADP+/NADPH balance and an increased demand for glutathione metabolism. Moreover, the nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key protective role in IDH1-mutated cells by prompting glutathione synthesis and ROS scavenging. Pharmacological inhibition of the Nrf2/glutathione pathway via brusatol administration exhibited a potent tumor suppressive effect on IDH1-mutated cancer in vitro and in vivo. Our findings highlight a possible therapeutic strategy that could be valuable for IDH1-mutated cancer treatment.
  15. Front Physiol. 2019 ;10 1140
    Wang J, Liu Y, Lian K, Shentu X, Fang J, Shao J, Chen M, Wang Y, Zhou M, Sun H.
      Recent studies show branched-chain amino acid (BCAA) catabolic pathway is defective in obese animals and humans, contributing to the pathogenesis of insulin resistance and diabetes. However, in the context of obesity, various processes including the dysfunctional lipid metabolism can affect insulin sensitivity and glycemic regulation. It remains unclear how BCAA catabolic defect may exert direct impacts on glucose metabolism without the disturbance of obesity. The current study characterized the glucose metabolism in lean mice in which the genetic deletion of PP2Cm leads to moderate BCAA catabolic defect. Interestingly, compared to the wildtype control, lean PP2Cm deficient mice showed enhanced insulin sensitivity and glucose tolerance, lower body weight, and the preference for carbohydrate over lipids utilization. Metabolomics profiling of plasma and tissues revealed significantly different metabolic patterns in the PP2Cm deficient mice, featured by the marked alterations in glucose metabolic processes, including gluconeogenesis/glycolysis, glycogen metabolism, and tricarboxylic acid cycle. The metabolic changes of glucose were predominantly observed in liver but not skeletal muscle or white adipose tissue. The elevated branched-chain keto acids (BCKAs) resulted from the BCAA catabolic defect may play a critical role in regulating the expression of key regulators of glucose metabolic processes and the activity of respiratory Complex II/succinate dehydrogenase in TCA cycle. Together, these results show BCAA catabolic defect significantly alters glucose metabolism in lean mice with some impacts different or even opposite from those in obese mice, highlighting the critical role of BCAA catabolism in glycemic regulation and the complex interplay between macronutrients in lean and obese animals.
    Keywords:  branched-chain amino acids; catabolic defect; glucose metabolism; lean mice; liver
  16. Mol Cell. 2019 Sep 16. pii: S1097-2765(19)30660-4. [Epub ahead of print]
    Su KH, Dai S, Tang Z, Xu M, Dai C.
      Through transcriptional control of the evolutionarily conserved heat shock, or proteotoxic stress, response, heat shock factor 1 (HSF1) preserves proteomic stability. Here, we show that HSF1, a physiological substrate for AMP-activated protein kinase (AMPK), constitutively suppresses this central metabolic sensor. By physically evoking conformational switching of AMPK, HSF1 impairs AMP binding to the γ subunits and enhances the PP2A-mediated de-phosphorylation, but it impedes the LKB1-mediated phosphorylation of Thr172, and retards ATP binding to the catalytic α subunits. These immediate and manifold regulations empower HSF1 to both repress AMPK under basal conditions and restrain its activation by diverse stimuli, thereby promoting lipogenesis, cholesterol synthesis, and protein cholesteroylation. In vivo, HSF1 antagonizes AMPK to control body fat mass and drive the lipogenic phenotype and growth of melanomas independently of its intrinsic transcriptional action. Thus, the physical AMPK-HSF1 interaction epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine.
    Keywords:  AMPK; HSF1; LKB1; SHH; SREBP1; cholesteroylation; conformational switch; lipogenesis; oncogenesis
  17. Nat Commun. 2019 Sep 27. 10(1): 4399
    Modi S, López-Doménech G, Halff EF, Covill-Cooke C, Ivankovic D, Melandri D, Arancibia-Cárcamo IL, Burden JJ, Lowe AR, Kittler JT.
      Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.
  18. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201903542. [Epub ahead of print]
    Valdor R, García-Bernal D, Riquelme D, Martinez CM, Moraleda JM, Cuervo AM, Macian F, Martinez S.
      The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own chaperone-mediated autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.
    Keywords:  chaperone-mediated autophagy; glioblastoma; immunosuppressive function; pericytes; tumor
  19. J Biol Chem. 2019 Sep 27. pii: jbc.RA119.010371. [Epub ahead of print]
    Newhardt MF, Batushansky A, Matsuzaki S, Young ZT, West M, Chin NC, Szweda LI, Kinter M, Humphries KM.
      The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Metabolic inflexibility, such as occurs with diabetes, increases cardiac reliance on fatty acids to meet energetic demands and this results in deleterious effects, including mitochondrial dysfunction, that contribute to pathophysiology. Enhancing glucose usage may mitigate metabolic inflexibility and be advantageous under such conditions. Here, we sought to identify how mitochondrial function and cardiac metabolism are affected in a transgenic mouse model of enhanced cardiac glycolysis (GlycoHi) basally and following a short-term (7d) high fat diet (HFD). GlycoHimice constitutively express an active form of phosphofructokinase-2, resulting in elevated levels of the PFK-1 allosteric activator fructose-2,6-bisphosphate. We report that basally GlycoHi mitochondria exhibit augmented pyruvate supported respiration relative to fatty acids. Nevertheless, both wild type and GlycoHi mitochondria had a similar shift towards increased rates of fatty acid supported respiration following HFD. Metabolic profiling by GC-MS revealed distinct features based on both genotype and diet, with a unique increase in branched chain amino acids in the GlycoHi HFD group. Targeted quantitative proteomics analysis also supported both genotype and diet-dependent changes in protein expression and uncovered an enhanced expression of pyruvate dehydrogenase kinase 4 (PDK4) in the GlycoHiHFD group.  These results support a newly identified mechanism whereby the levels of fructose-2,6-bisphosphate promote mitochondrial PDK4 levels and identifies a secondary adaptive response that prevents excessive mitochondrial pyruvate oxidation when glycolysis is sustained after a high fat dietary challenge.
    Keywords:  branched chain amino acids; cardiac metabolism; glycolysis; mitochondria; phosphofructokinase; proteomics
  20. Methods Appl Fluoresc. 2019 Sep 25.
    V Chacko J, Eliceiri KW.
      Autofluorescence based fluorescence lifetime imaging microscopy (AF-FLIM) techniques have come a long way from early studies on cancer characterization and have now been widely employed in several cellular and animal studies covering a wide range of diseases. The majority of research in autofluorescence imaging (AFI) study metabolic fluxes in live biological samples. However, tissues from clinical or scientific studies are often chemically fixed for preservation and stabilization of tissue morphology. Fixation is particularly crucial for enzymatic, functional, or histopathology studies. Interpretations of metabolic imaging such as optical redox intensity imaging and AF-FLIM, have often been viewed as potentially unreliable in a fixed sample due to lack of studies in this field. In this study, we carefully evaluate the possibility of extracting microenvironment information in fixed tissues using reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) endogenous fluorescence. The ability to distinguish changes such as metabolism and pH using intrinsic fluorescence in fixed tissues has great pathological value. In this work, we show that the lifetime based metabolic contrast in a sample is preserved after chemical fixation. The fluorescence lifetime of a sample increases with an additive fixative like formaldehyde; however, the fixed tissues retain metabolic signatures even after fixation. This study presents an opportunity to successfully image archived unstained histopathology tissues, and generate useful AF-FLIM signatures. We demonstrate the capability to draw metabolic interpretations in fixed tissues even after long periods of storage.
    Keywords:  Autofluorescence Imaging; Effects of Fixation; FLIM; Metabolic Imaging; NAD(P)H FLIM; NADH Autofluorescence
  21. Sci Signal. 2019 Sep 24. pii: eaaw8288. [Epub ahead of print]12(600):
    McFall T, Diedrich JK, Mengistu M, Littlechild SL, Paskvan KV, Sisk-Hackworth L, Moresco JJ, Shaw AS, Stites EC.
      Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient's tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRASG13D) behaves differently; for unknown reasons, KRASG13D CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.
  22. J Clin Invest. 2019 Sep 23. pii: 131931. [Epub ahead of print]
    Sarabhai T, Roden M.
      Fasting requires complex endocrine and metabolic interorgan crosstalk, which involves shifting from glucose to fatty acid oxidation, derived from adipose tissue lipolysis, in order to preserve glucose for the brain. The glucose-alanine (Cahill) cycle is critical for regenerating glucose. In this issue of JCI, Petersen et al. report on their use of an innovative stable isotope tracer method to show that skeletal muscle-derived alanine becomes rate controlling for hepatic mitochondrial oxidation and, in turn, for glucose production during prolonged fasting. These results provide new insight into skeletal muscle-liver metabolic crosstalk during the fed-to-fasting transition in humans.
  23. J Biol Chem. 2019 Sep 27. pii: jbc.RA119.010185. [Epub ahead of print]
    Zheng F, Jia B, Dong F, Liu L, Rasul F, He J, Fu C.
      Mitochondria undergo morphological and dynamic changes in response to environmental stresses. Few studies have focused on addressing mitochondrial remodeling under stress. Using the fission yeast Schizosaccharomyces pombe as a model organism, here we investigated mitochondrial remodeling under glucose starvation. We employed live-cell microscopy to monitor mitochondrial morphology and dynamics of cells in profusion chambers under glucose starvation. Our results revealed that mitochondria fragment within minutes after glucose starvation and that the dynamin GTPase Dnm1 is required for promoting the mitochondrial fragmentation. Moreover, we found that glucose starvation enhances Dnm1 localization to mitochondria and increases the frequency of mitochondrial fission, but decreases protein kinase A (PKA) activity. We further demonstrate that low PKA activity enhances glucose starvation-induced mitochondrial fragmentation, whereas high PKA activity confers resistance to glucose starvation-induced mitochondrial fragmentation. Moreover, we observed that AMP-activated protein kinase (AMPK) is not involved in regulating mitochondrial fragmentation under glucose starvation. Of note, Glucose starvation-induced mitochondrial fragmentation was associated with enhanced reactive oxygen species (ROS) production. Our work provides detailed mechanistic insights into mitochondrial remodeling in response to glucose starvation.
    Keywords:  dynamin; fungi; glucose; mitochondria; stress
  24. Cell Death Dis. 2019 Sep 24. 10(10): 710
    Wu W, Zhao D, Shah SZA, Zhang X, Lai M, Yang D, Wu X, Guan Z, Li J, Zhao H, Li W, Gao H, Zhou X, Qiao J, Yang L.
      Prion diseases caused by the cellular prion protein (PrPC) conversion into a misfolded isoform (PrPSc) are associated with multiple mitochondrial damages. We previously reported mitochondrial dynamic abnormalities and cell death in prion diseases via modulation of a variety of factors. Optic atrophy 1 (OPA1) is one of the factors that control mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergetics, and cristae integrity. In this study, we observed downregulation of OPA1 in prion disease models in vitro and in vivo, mitochondria structure damage and dysfunction, loss of mtDNA, and neuronal apoptosis. Similar mitochondria findings were seen in OPA1-silenced un-infected primary neurons. Overexpression of OPA1 not only alleviated prion-induced mitochondrial network fragmentation and mtDNA loss, decrease in intracellular ATP, increase in ADP/ATP ratio, and decrease in mitochondrial membrane potential but also protected neurons from apoptosis by suppressing the release of cytochrome c from mitochondria to cytosol and activation of the apoptotic factor, caspase 3. Our results demonstrated that overexpression of OPA1 alleviates prion-associated mitochondrial network fragmentation and cristae remodeling, mitochondrial dysfunction, mtDNA depletion, and neuronal apoptosis, suggesting that OPA1 may be a novel and effective therapeutic target for prion diseases.
  25. Nat Cell Biol. 2019 Sep 23.
    Lim CY, Davis OB, Shin HR, Zhang J, Berdan CA, Jiang X, Counihan JL, Ory DS, Nomura DK, Zoncu R.
      Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.
  26. Nat Cell Biol. 2019 Sep 23.
    Di Giammartino DC, Kloetgen A, Polyzos A, Liu Y, Kim D, Murphy D, Abuhashem A, Cavaliere P, Aronson B, Shah V, Dephoure N, Stadtfeld M, Tsirigos A, Apostolou E.
      Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
  27. Cell Rep. 2019 Sep 24. pii: S2211-1247(19)31097-6. [Epub ahead of print]28(13): 3329-3337.e5
    Wiley CD, Liu S, Limbad C, Zawadzka AM, Beck J, Demaria M, Artwood R, Alimirah F, Lopez-Dominguez JA, Kuehnemann C, Danielson SR, Basisty N, Kasler HG, Oron TR, Desprez PY, Mooney SD, Gibson BW, Schilling B, Campisi J, Kapahi P.
      Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.
    Keywords:  SASP; aging; cellular senescence; chemotherapy; clotting; coagulation; homeostasis; proteomics; secretion; thrombosis
  28. Nat Rev Nephrol. 2019 Sep 25.
    van Swelm RPL, Wetzels JFM, Swinkels DW.
      Iron is an essential element that is indispensable for life. The delicate physiological body iron balance is maintained by both systemic and cellular regulatory mechanisms. The iron-regulatory hormone hepcidin assures maintenance of adequate systemic iron levels and is regulated by circulating and stored iron levels, inflammation and erythropoiesis. The kidney has an important role in preventing iron loss from the body by means of reabsorption. Cellular iron levels are dependent on iron import, storage, utilization and export, which are mainly regulated by the iron response element-iron regulatory protein (IRE-IRP) system. In the kidney, iron transport mechanisms independent of the IRE-IRP system have been identified, suggesting additional mechanisms for iron handling in this organ. Yet, knowledge gaps on renal iron handling remain in terms of redundancy in transport mechanisms, the roles of the different tubular segments and related regulatory processes. Disturbances in cellular and systemic iron balance are recognized as causes and consequences of kidney injury. Consequently, iron metabolism has become a focus for novel therapeutic interventions for acute kidney injury and chronic kidney disease, which has fuelled interest in the molecular mechanisms of renal iron handling and renal injury, as well as the complex dynamics between systemic and local cellular iron regulation.
  29. Cancer Discov. 2019 Sep 27.
      Restoration of p53 led to an accumulation of αKG in mouse pancreatic cancer cells.
  30. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201901759. [Epub ahead of print]
    Kolitsida P, Zhou J, Rackiewicz M, Nolic V, Dengjel J, Abeliovich H.
      Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.
    Keywords:  Saccharomyces cerevisiae; autophagy; mitophagy; phosphatase; protein phosphorylation
  31. J Clin Invest. 2019 Sep 24. pii: 128514. [Epub ahead of print]
    Del Dotto V, Ullah F, Di Meo I, Magini P, Gusic M, Maresca A, Caporali L, Palombo F, Tagliavini F, Baugh EH, Macao B, Szilagyi Z, Péron C, Gustafson MA, Khan K, La Morgia C, Barboni P, Carbonelli M, Valentino ML, Liguori R, Shashi V, Sullivan JA, Nagaraj S, El-Dairi M, Iannaccone A, Cutcutache I, Bertini E, Carrozzo R, Emma F, Diomedi-Camassei F, Zanna C, Armstrong M, Page MJ, Boesch S, Wortmann SB, Kopajtich R, Stong N, Sperl W, Davis E, Copeland WC, Seri M, Falkenberg M, Prokisch H, Katsanis N, Tiranti V, Pippucci T, Carelli V.
      Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single strand binding protein (SSBP1) in four families with dominant and one with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect its amount and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids and 7S-DNA amounts as well as mtDNA replication, impacting replisome machinery. The variable mtDNA depletion in cells reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits and complexes amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex-vivo in biopsies of affected tissues, like kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by wild-type mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as cause of human pathology.
    Keywords:  Bioenergetics; Genetic diseases; Genetics; Mitochondria; Ophthalmology
  32. J Clin Invest. 2019 Sep 23. pii: 129913. [Epub ahead of print]
    Petersen KF, Dufour S, Cline GW, Shulman GI.
      In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-13C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.
    Keywords:  Amino acid metabolism; Endocrinology; Fatty acid oxidation; Intermediary metabolism; Metabolism
  33. Sci Rep. 2019 Sep 25. 9(1): 13898
    Weinrich TW, Kam JH, Ferrara BT, Thompson EP, Mitrofanis J, Jeffery G.
      Mitochondria provide energy for cellular function. We examine daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity. These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health.
  34. J Clin Invest. 2019 Sep 24. pii: 128513. [Epub ahead of print]
    Piro-Mégy C, Sarzi E, Tarrés-Solé A, Péquignot M, Hensen F, Quilès M, Manes G, Chakraborty A, Sénéchal A, Bocquet B, Cazevieille C, Roubertie A, Müller A, Charif M, Goudenège D, Lenaers G, Wilhelm H, Kellner U, Weisschuh N, Wissinger B, Zanlonghi X, Hamel C, Spelbrink JN, Solà M, Delettre C.
      Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDS), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in SSBP1 in five unrelated families, leading to the R38Q and R107Q amino-acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a new revised SSBP1 crystal structure. Structural analysis suggests that both mutations affect dimer interactions and presumably distort the DNA binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication and induces mtDNA depletion. Our study, showing that mutations in SSBP1 cause a novel form of dominant optic atrophy frequently accompanied with foveopathy, brings new insights into mtDNA maintenance disorders.
    Keywords:  Genetics; Mitochondria; Neurodegeneration; Ophthalmology
  35. Nature. 2019 Sep 25.
    Findeisen M, Allen TL, Henstridge DC, Kammoun H, Brandon AE, Baggio LL, Watt KI, Pal M, Cron L, Estevez E, Yang C, Kowalski GM, O'Reilly L, Egan C, Sun E, Thai LM, Krippner G, Adams TE, Lee RS, Grötzinger J, Garbers C, Risis S, Kraakman MJ, Mellet NA, Sligar J, Kimber ET, Young RL, Cowley MA, Bruce CR, Meikle PJ, Baldock PA, Gregorevic P, Biden TJ, Cooney GJ, Keating DJ, Drucker DJ, Rose-John S, Febbraio MA.
      The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.