bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒08‒29
forty-five papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. J Vis Exp. 2021 Aug 06.
      Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.
  2. Biomolecules. 2021 Aug 09. pii: 1177. [Epub ahead of print]11(8):
      Cancer cell culture is routinely performed under superphysiologic O2 levels and in media such as Dulbecco's Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O2 levels in cell culture. Given these observations, we investigated how media composition and O2 levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O2 levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O2 levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity.
    Keywords:  cell culture; glycolysis; metabolism; mitochondria; mitochondrial networks; oxidative phosphorylation
  3. J Biochem Mol Toxicol. 2021 Aug 25. e22898
      Maintenance of mitochondrial oxidative phosphorylation capacity and other mitochondrial functions are essential for the prevention of mitochondrial dysfunction-related diseases such as neurodegenerative, cardiovascular, and liver diseases. To date, no well-known treatment modality has been developed to prevent or reduce mitochondrial dysfunction. However, a novel approach that transplants fully functional mitochondria directly into defective cells has recently caught the attention of scientists. In this review, we provide an overview of the cell/tissue source of the mitochondria to prompt cell regeneration or tissue repair in vitro and in vivo applications. The animal and human models entail that effective procedures should be used in the isolation and confirmation of mitochondrial membrane potential and function. We believe that these procedures for mitochondrial transplantation for tissue or cell culture will confirm intact, viable, and free from contamination isolated mitochondria from the appropriate sources.
    Keywords:  mitochondria dysfunction; mitochondrial isolation; mitochondrial transplantation
  4. Biomolecules. 2021 Aug 09. pii: 1178. [Epub ahead of print]11(8):
      2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.
    Keywords:  artificial membranes; membrane potential; mitochondrial uncoupler; molecular dynamics simulations; proton conductance; protonophore
  5. J Lipid Res. 2021 Aug 24. pii: S0022-2275(21)00093-6. [Epub ahead of print] 100111
      The molecular assembly of cells depends not only on the balance between anabolism and catabolism, but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum- and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favours the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory Complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research.
    Keywords:  Cardiolipin; Fatty acids; Lipids; Mass spectroscopy; Mitochondria
  6. Cell Chem Biol. 2021 Aug 24. pii: S2451-9456(21)00365-2. [Epub ahead of print]
      Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. In vitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.
    Keywords:  DNA alkylation; DNA mutation; designer small molecule; heteroplasmy; mitochondria; mitochondrial DNA; mitochondrial disease; pyrrole-imidazole polyamide
  7. Nutrients. 2021 Jul 24. pii: 2533. [Epub ahead of print]13(8):
      Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.
    Keywords:  brain; ketogenic diet; liver; mitochondria; skeletal muscle
  8. FEBS J. 2021 Aug 24.
      Coenzyme Q (CoQ, ubiquinone) is the electron-carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH-dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron-transferring flavoprotein dehydrogenase, mitochondrial glycerol-3-phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ-linked metabolism depends on CoQ re-oxidation by the mitochondrial complex III (cytochrome bc1 complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ-linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ-dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation.
    Keywords:  coenzyme Q; complex III; mitochondrial disease; oxidative phosphorylation; ubiquinone
  9. Cell Rep. 2021 Aug 24. pii: S2211-1247(21)00999-2. [Epub ahead of print]36(8): 109565
      Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.
    Keywords:  Drp1; brown adipose tissue; insulin resistance; metabolic syndrome; mitochondrial dynamics; mitochondrial respiration; phosphorylation; thermoneutrality
  10. STAR Protoc. 2021 Sep 17. 2(3): 100735
      Depending on metabolic requirements, skeletal muscle mitochondria integrate O2 consumption and ATP production with lipid, glucose, or amino acid metabolism. Free fatty acids (FFAs) are the main source of energy during rest and mild-intensity exercise. We present a detailed protocol for measuring FFA-β-oxidation coupled with O2 respiration by a Clark-type electrode in isolated mitochondria from mouse soleus oxidative muscle. We optimized the procedure, including buffer composition, protease treatment, and quantifiable parameters (P/O, Phosphate/Oxygen Ratio; OCR, Oxygen Consumption Rate; RCR,Respiration Control Rate; OSR, Oligomycin Sensitive Respiration). For complete details on the use and execution of this protocol, please refer to Sanchez-Gonzalez et al. (2020).
    Keywords:  cell biology; cell isolation; cell membrane; cell separation/fractionation; metabolism
  11. Cancers (Basel). 2021 Aug 18. pii: 4151. [Epub ahead of print]13(16):
      Colorectal cancer is the second most common cancer and the third cancer-associated death in Taiwan. Currently used serum markers for detecting colorectal cancer lack excellent diagnostic accuracy, which results in colorectal cancer being often recognized too late for successful therapy. Mitophagy is the selective autophagic degradation of damaged or excessive mitochondria. DJ-1 is an antioxidant protein that attenuates oxidative stress and maintains mitochondrial quality through activating mitophagy. Mitophagy activation contributes to anti-cancer drug resistance. However, the role of DJ-1-induced mitophagy in colorectal cancer progression remains unclear. In the present study, we collected matched tumor and adjacent normal tissues and serum from patients and cancer cells to demonstrate the clinical value and physiological function of DJ-1 in colorectal cancer. We found that DJ-1 increased in tumor tissues and serum; it was positively correlated with TNM (tumor-node-metastasis) stages of colorectal cancer patients. Through stable knockdown DJ-1 expression in metastatic colorectal adenocarcinoma cells SW620, DJ-1 knockdown inhibited cancer cell survival, migration, and colony formation. In SW620 cells, DJ-1 knockdown induced an incomplete autophagic response that did not affect ATP production; DJ-1 knockdown enhanced intracellular reactive oxygen species generation and damaged mitochondrial accumulation and mitophagy inhibition. It suggests that DJ-1 knockdown inhibits mitophagy that causes metastatic colorectal adenocarcinoma cells to be unable to remove damaged mitochondria and further enhance cancer cell apoptosis. Our data indicate that DJ-1 might be clinically valuable as serum and tissue biomarkers for predicting the TNM stage in colorectal cancer patients. Since DJ-1-induced mitophagy promotes tumor progression, DJ-1 inhibition is a potential therapeutic strategy for colorectal cancer treatment.
    Keywords:  DJ-1; autophagy; biomarker; colorectal cancer; mitophagy
  12. Mol Biol Cell. 2021 Aug 25. mbcE20070457
      The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multi-subunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress is not sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8 is a bonafide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggest a universal role of MRX8 family of proteins in regulating mitochondrial function.
  13. Cell Stem Cell. 2021 Aug 21. pii: S1934-5909(21)00296-4. [Epub ahead of print]
      The electron transport chain promotes aspartate synthesis, which is required for cancer cell proliferation. However, it is unclear whether aspartate is limiting in normal stem cells. We found that mouse hematopoietic stem cells (HSCs) depend entirely on cell-autonomous aspartate synthesis, which increases upon HSC activation. Overexpression of the glutamate/aspartate transporter, Glast, or deletion of glutamic-oxaloacetic transaminase 1 (Got1) each increased aspartate levels in HSCs/progenitor cells and increased the function of HSCs but not colony-forming progenitors. Conversely, deletion of Got2 reduced aspartate levels and the function of HSCs but not colony-forming progenitors. Deletion of Got1 and Got2 eliminated HSCs. Isotope tracing showed aspartate was used to synthesize asparagine and purines. Both contributed to increased HSC function as deletion of asparagine synthetase or treatment with 6-mercaptopurine attenuated the increased function of GLAST-overexpressing HSCs. HSC function is thus limited by aspartate, purine, and asparagine availability during hematopoietic regeneration.
    Keywords:  asparagine; aspartate; electron transport chain; hematopoietic stem cell; metabolism; mitochondria; purine; regeneration
  14. Physiol Rep. 2021 Aug;9(16): e15016
      This study aimed to examine the effects of voluntary wheel running on cancer cachexia-induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4-hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia.
    Keywords:  cancer cachexia; mitochondria; oxidative stress; skeletal muscle; voluntary exercise
  15. Cancers (Basel). 2021 Aug 09. pii: 4009. [Epub ahead of print]13(16):
      The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.
    Keywords:  folate; glycine; one-carbon metabolism; serine; serine hydroxymethyltransferase
  16. Front Mol Biosci. 2021 ;8 711436
      Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome c oxidase (CcO). This converts the energy released in the cytochrome c/oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis. Despite detailed knowledge of electron and proton transfer paths, a central question remains as to whether the coupling between electron and proton transfer in mammalian mitochondrial forms of CcO is mechanistically equivalent to its bacterial counterparts. Here, we focus on the conserved span between H376 and G384 of transmembrane helix (TMH) X of subunit I. This conformationally-dynamic section has been suggested to link the redox activity with the putative H pathway of proton transfer in mammalian CcO. The two helix X mutants, Val380Met (V380M) and Gly384Asp (G384D), generated in the genetically-tractable yeast CcO, resulted in a respiratory-deficient phenotype caused by the inhibition of intra-protein electron transfer and CcO turnover. Molecular aspects of these variants were studied by long timescale atomistic molecular dynamics simulations performed on wild-type and mutant bovine and yeast CcOs. We identified redox- and mutation-state dependent conformational changes in this span of TMH X of bovine and yeast CcOs which strongly suggests that this dynamic module plays a key role in optimizing intra-protein electron transfers.
    Keywords:  density functional theory; mitochondrial respiration; molecular dynamics simulations; proton pumping; yeast bioenergetics
  17. FEBS J. 2021 Aug 26.
      ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signaling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Finally, we will explore the palette of genetically-encoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.
    Keywords:  ATP; ATP synthase; biochemical assays; fluorescence-based tools; mitochondria
  18. J Biol Chem. 2021 Aug 19. pii: S0021-9258(21)00904-2. [Epub ahead of print] 101101
      The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/- mice. Relative to wild type (WT) controls, Rdh10+/- males fed a high-fat diet decrease reliance on fatty-acid oxidation, and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/- females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/- male GM decrease 38% relative to WT. Rdh10+/- male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/- female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.
    Keywords:  ATP; Rdh10; cytochrome c oxidase (Complex IV); estrogen; gene knockout; metabolic regulation; retinoic acid; running endurance; skeletal muscle; vitamin A
  19. Metabolites. 2021 Jul 26. pii: 480. [Epub ahead of print]11(8):
      The biguanide drug metformin has been widely used for the treatment of type 2 diabetes, and there is evidence supporting the anticancer effect of metformin despite some controversy. Here, we report the growth inhibitory activity of metformin in the breast cancer (MCF-7) cells, both in vitro and in vivo, and the associated metabolic changes. In particular, a decrease in a well-known oncometabolite 2-hydroxyglutarate (2-HG) was discovered by a metabolomics approach. The decrease in 2-HG by metformin was accompanied by the reduction in histone methylation, consistent with the known tumorigenic mechanism of 2-HG. The relevance of 2-HG inhibition in breast cancer was also supported by a higher level of 2-HG in human breast cancer tissues. Genetic knockdown of PHGDH identified the PHGDH pathway as the producer of 2-HG in the MCF-7 cells that do not carry isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) mutations, the conventional producer of 2-HG. We also showed that metformin's inhibitory effect on the PHGDH-2HG axis may occur through the regulation of the AMPK-MYC pathway. Overall, our results provide an explanation for the coherent pathway from complex I inhibition to epigenetic changes for metformin's anticancer effect.
    Keywords:  2-HG; PHGDH; anticancer effect; metabolomics; metformin
  20. Int J Mol Sci. 2021 Aug 06. pii: 8468. [Epub ahead of print]22(16):
      In the human heart, the energy supplied by the production of ATP is predominately accomplished by ß-oxidation in mitochondria, using fatty acids (FAs) as the primary fuel. Long-chain acylcarnitines (LCACs) are intermediate forms of FA transport that are essential for FA delivery from the cytosol into mitochondria. Here, we analyzed the impact of the LCACs C18 and C18:1 on mitochondrial function and, subsequently, on heart functionality in the in vivo vertebrate model system of zebrafish (Danio rerio). Since LCACs are formed and metabolized in mitochondria, we assessed mitochondrial morphology, structure and density in C18- and C18:1-treated zebrafish and found no mitochondrial alterations compared to control-treated (short-chain acylcarnitine, C3) zebrafish embryos. However, mitochondrial function and subsequently ATP production was severely impaired in C18- and C18:1-treated zebrafish embryos. Furthermore, we found that C18 and C18:1 treatment of zebrafish embryos led to significantly impaired cardiac contractile function, accompanied by reduced heart rate and diminished atrial and ventricular fractional shortening, without interfering with cardiomyocyte differentiation, specification and growth. In summary, our findings provide insights into the direct role of long-chain acylcarnitines on vertebrate heart function by interfering with regular mitochondrial function and thereby energy allocation in cardiomyocytes.
    Keywords:  cardiovascular disease; long-chain acylcarnitine; mitochondria; zebrafish
  21. Int J Mol Sci. 2021 Aug 09. pii: 8560. [Epub ahead of print]22(16):
      The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5-2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.
    Keywords:  NAD+; NADH; calcium retention capacity; cytosolic; external regulatory site; permeability transition pore; pore closure
  22. Cell Metab. 2021 Aug 17. pii: S1550-4131(21)00365-X. [Epub ahead of print]
      Adipocytes undergo intense energetic stress in obesity resulting in loss of mitochondrial mass and function. We have found that adipocytes respond to mitochondrial stress by rapidly and robustly releasing small extracellular vesicles (sEVs). These sEVs contain respiration-competent, but oxidatively damaged mitochondrial particles, which enter circulation and are taken up by cardiomyocytes, where they trigger a burst of ROS. The result is compensatory antioxidant signaling in the heart that protects cardiomyocytes from acute oxidative stress, consistent with a preconditioning paradigm. As such, a single injection of sEVs from energetically stressed adipocytes limits cardiac ischemia/reperfusion injury in mice. This study provides the first description of functional mitochondrial transfer between tissues and the first vertebrate example of "inter-organ mitohormesis." Thus, these seemingly toxic adipocyte sEVs may provide a physiological avenue of potent cardio-protection against the inevitable lipotoxic or ischemic stresses elicited by obesity.
    Keywords:  adipocyte; cardiovascular disease; diabetes; exosomes; extracellular vesicles; mitochondria; mitochondrial dysfunction; mitohormesis; obesity; stress response
  23. Eur J Hum Genet. 2021 Aug 23.
      In a Dutch non-consanguineous patient having mitochondrial encephalomyopathy with complex I and complex IV deficiency, whole exome sequencing revealed two compound heterozygous variants in SLIRP. SLIRP gene encodes a stem-loop RNA-binding protein that regulates mitochondrial RNA expression and oxidative phosphorylation (OXPHOS). A frameshift and a deep-intronic splicing variant reduced the amount of functional wild-type SLIRP RNA to 5%. Consequently, in patient fibroblasts, MT-ND1, MT-ND6, and MT-CO1 expression was reduced. Lentiviral transduction of wild-type SLIRP cDNA in patient fibroblasts increased MT-ND1, MT-ND6, and MT-CO1 expression (2.5-7.2-fold), whereas mutant cDNAs did not. A fourfold decrease of citrate synthase versus total protein ratio in patient fibroblasts indicated that the resulting reduced mitochondrial mass caused the OXPHOS deficiency. Transduction with wild-type SLIRP cDNA led to a 2.4-fold increase of this ratio and partly restored OXPHOS activity. This confirmed causality of the SLIRP variants. In conclusion, we report SLIRP variants as a novel cause of mitochondrial encephalomyopathy with OXPHOS deficiency.
  24. Carcinogenesis. 2021 Aug 27. pii: bgab079. [Epub ahead of print]
      XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in XP-C cells (XPC-null) compared to an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, while AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine ​​(NAC) increased glutathione concentration, decreased basal H2O2 production, p53 levels and sensitivity to AA treatment in the XPC-null back to the levels found in XPC-wt cells. Thus, the results suggest a critical role for mitochondrially-generated H2O2 in the regulation of p53 expression, which in turn modulates XP-C sensitivity to agents that cause mitochondrial stress.
    Keywords:  XPC; antimycin A; hydrogen peroxide; mitochondria; p53
  25. Sci Adv. 2021 Aug;pii: eabg7287. [Epub ahead of print]7(35):
      Changes in metabolism that affect mitochondrial and glycolytic networks are hallmarks of cancer, but their impact in disease is still elusive. Using global proteomics and ubiquitome screens, we now show that Parkin, an E3 ubiquitin ligase and key effector of mitophagy altered in Parkinson's disease, shuts off mitochondrial dynamics and inhibits the non-oxidative phase of the pentose phosphate pathway. This blocks tumor cell movements, creates metabolic and oxidative stress, and inhibits primary and metastatic tumor growth. Uniformly down-regulated in cancer patients, Parkin tumor suppression requires its E3 ligase function, is reversed by antioxidants, and is independent of mitophagy. These data demonstrate that cancer metabolic networks are potent oncogenes directly targeted by endogenous tumor suppression.
  26. Nat Med. 2021 Aug 23.
      Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis.
  27. Diabetologia. 2021 Aug 27.
      AIMS/HYPOTHESIS: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (NntTr [Tr, truncated]) is usually considered to be the main cause of the lower glucose tolerance of C57BL/6J vs C57BL/6N mice. However, the impact of this mutation on glucose tolerance remains disputed. Here, we singled out the impact of NntTr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (EGSH), glucose-stimulated insulin secretion (GSIS) and glucose tolerance.METHODS: Male and female N5BL/6J mice that express wild-type Nnt (NntWT) or NntTr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J NntWT/Tr heterozygous mice. C57BL/6J and C57BL/6N mice were from Janvier Labs. The Nnt genotype was confirmed by PCR and the genetic background by whole genome sequencing of one mouse of each type. Glucose tolerance was assessed by IPGTT, ITT and fasting/refeeding tests. Stimulus-secretion coupling events and GSIS were measured in isolated pancreatic islets. Cytosolic and mitochondrial EGSH were measured using the fluorescent redox probe GRX1-roGFP2 (glutaredoxin 1 fused to redox-sensitive enhanced GFP).
    RESULTS: The Nnt genotype and genetic background of each type of mouse were confirmed. As reported previously in C57BL/6N vs C57BL/6J islets, the glucose regulation of mitochondrial (but not cytosolic) EGSH and of NAD(P)H autofluorescence was markedly improved in N5-WT vs N5-Tr islets, confirming the role of NNT in mitochondrial redox regulation. However, ex vivo GSIS was only 1.2-1.4-times higher in N5-WT vs N5-Tr islets, while it was 2.4-times larger in C57BL/6N vs N5-WT islets, questioning the role of NNT in GSIS. In vivo, the ITT results did not differ between N5-WT and N5-Tr or C57BL/6N mice. However, the glucose excursion during an IPGTT was only 15-20% lower in female N5-WT mice than in N5-Tr and C57BL/6J mice and remained 3.5-times larger than in female C57BL/6N mice. Similar observations were made during a fasting/refeeding test. A slightly larger (~30%) impact of NNT on glucose tolerance was found in males.
    CONCLUSIONS/INTERPRETATION: Although our results confirm the importance of NNT in the regulation of mitochondrial redox state by glucose, they markedly downsize the role of NNT in the alteration of GSIS and glucose tolerance in C57BL/6J vs C57BL/6N mice. Therefore, documenting an NntWT genotype in C57BL/6 mice does not provide proof that their glucose tolerance is as good as in C57BL/6N mice.
    Keywords:  Genetic background; Glucose homeostasis; Glutathione redox state; Insulin secretion; Mitochondria; NADPH
  28. Front Immunol. 2021 ;12 714897
      Psoriasis is a common immune-mediated, chronic, inflammatory skin disease that affects approximately 2-3% of the population worldwide. Although there is increasing evidence regarding the essential roles of the interleukin (IL)-23/IL-17 axis and dendritic cell (DC)-T cell crosstalk in the development of skin inflammation, the contributions of mitochondrial function to psoriasis are unclear. In a mouse model of imiquimod (IMQ)-induced psoriasiform skin inflammation, we found that hematopoietic cell-specific genetic deletion of p32/C1qbp, a regulator of mitochondrial protein synthesis and metabolism, protects mice from IMQ-induced psoriatic inflammation. Additionally, we demonstrate that p32/C1qbp is an important regulator of IMQ-induced DC activation, both in vivo and in vitro. We also found that p32/C1qbp-deficient DCs exhibited impaired production of IL-1β, IL-23, and mitochondrial reactive oxygen species (mtROS) after IMQ stimulation. Because the inhibition of mtROS suppressed IMQ-induced DC activation and psoriatic inflammation, we presume that p32/C1qbp and mtROS can serve as therapeutic targets in psoriasis.
    Keywords:  C1qbp/p32; IL-1β; dendritic cells; mitochondrial ROS; psoriasis
  29. EMBO J. 2021 Aug 23. e107988
      The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.
    Keywords:  POLRMT; PolG; mitochondrial replication; mitochondrial transcription; primosome
  30. J Biol Chem. 2021 Aug 18. pii: S0021-9258(21)00903-0. [Epub ahead of print] 101100
      mTOR complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. While considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 (mTORC1) but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at alkaline pH induces a rapid rise in pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and -survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi attenuates mTOR- and AMPK-mediated apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK-mTORC2 and promote cell survival. As elevated pHi represents an under-appreciated hallmark of cancer cells, we propose that alkaline pHi stress sensing by AMPK-mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.
    Keywords:  AMPK; Akt; intracellular pH (pHi); mTORC2
  31. Nat Commun. 2021 08 24. 12(1): 5103
      Hypercholesterolemia and dyslipidemia are associated with an increased risk for many cancer types and with poor outcomes in patients with established disease. Whereas the mechanisms by which this occurs are multifactorial we determine that chronic exposure of cells to 27-hydroxycholesterol (27HC), an abundant circulating cholesterol metabolite, selects for cells that exhibit increased cellular uptake and/or lipid biosynthesis. These cells exhibit substantially increased tumorigenic and metastatic capacity. Notably, the metabolic stress imposed upon cells by the accumulated lipids requires sustained expression of GPX4, a negative regulator of ferroptotic cell death. We show that resistance to ferroptosis is a feature of metastatic cells and further demonstrate that GPX4 knockdown attenuates the enhanced tumorigenic and metastatic activity of 27HC resistant cells. These findings highlight the general importance of ferroptosis in tumor growth and metastasis and suggest that dyslipidemia/hypercholesterolemia impacts cancer pathogenesis by selecting for cells that are resistant to ferroptotic cell death.
  32. Int J Mol Sci. 2021 Aug 13. pii: 8688. [Epub ahead of print]22(16):
      Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.
    Keywords:  acute lymphoblastic leukemia; calcium overload; cannabidiol; cyclophilin D; mitochondria; mitochondrial permeability transition pore; tamoxifen
  33. J Hepatol. 2021 Aug 24. pii: S0168-8278(21)02004-3. [Epub ahead of print]
      BACKGROUND&AIMS: Patients with acute decompensation (AD) of cirrhosis progressing to acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from AD patients with and without ACLF.METHODS: The study included samples from AD patients (108 without and 128 with ACLF) and 41 healthy subjects. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array.
    RESULTS: Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In ACLF patients, mitochondrial function analysis uncovered two break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism.
    CONCLUSIONS: Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF.
    LAY SUMMARY: Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as the consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.
    Keywords:  ACLF; RNA-seq; acute decompensated cirrhosis; immune cells; metabolic phenotype; mitochondria
  34. Nucleic Acids Res. 2021 Aug 24. pii: gkab726. [Epub ahead of print]
      Diagnosing mitochondrial disorders remains challenging. This is partly because the clinical phenotypes of patients overlap with those of other sporadic and inherited disorders. Although the widespread availability of genetic testing has increased the rate of diagnosis, the combination of phenotypic and genetic heterogeneity still makes it difficult to reach a timely molecular diagnosis with confidence. An objective, systematic method for describing the phenotypic spectra for each variant provides a potential solution to this problem. We curated the clinical phenotypes of 6688 published individuals with 89 pathogenic mitochondrial DNA (mtDNA) mutations, collating 26 348 human phenotype ontology (HPO) terms to establish the MitoPhen database. This enabled a hypothesis-free definition of mtDNA clinical syndromes, an overview of heteroplasmy-phenotype relationships, the identification of under-recognized phenotypes, and provides a publicly available reference dataset for objective clinical comparison with new patients using the HPO. Studying 77 patients with independently confirmed positive mtDNA diagnoses and 1083 confirmed rare disease cases with a non-mitochondrial nuclear genetic diagnosis, we show that HPO-based phenotype similarity scores can distinguish these two classes of rare disease patients with a false discovery rate <10% at a sensitivity of 80%. Enriching the MitoPhen database with more patients will improve predictions for increasingly rare variants.
  35. MethodsX. 2021 ;8 101197
      We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles. Optimum conditions with respect to detergent concentration, time, sample size, and yield are discussed. The method's utility in downstream analyses and ease of processing multiple samples simultaneously is also described. All the reagents in this method can be assembled in-house, are economical, and are comparable, if not superior, to commercially available kits in terms of mitochondrial yield and integrity. • Rapid enrichment of mitochondria from mammalian cells using commonly available reagents. • Multiple samples can be processed simultaneously. • Works over a wide range of sample size (1 million to 100 million cells).
    Keywords:  Chemical lysis of cells; Mitochondria purification; Purity and integrity of mitochondria; Subcellular fractionation
  36. Nat Cancer. 2021 Aug;2(8): 853-864
      Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.
  37. Nat Commun. 2021 Aug 26. 12(1): 5137
      Serial circulating tumor DNA (ctDNA) monitoring is emerging as a non-invasive strategy to predict and monitor immune checkpoint blockade (ICB) therapeutic efficacy across cancer types. Yet, limited data exist to show the relationship between ctDNA dynamics and tumor genome and immune microenvironment in patients receiving ICB. Here, we present an in-depth analysis of clinical, whole-exome, transcriptome, and ctDNA profiles of 73 patients with advanced solid tumors, across 30 cancer types, from a phase II basket clinical trial of pembrolizumab (NCT02644369) and report changes in genomic and immune landscapes (primary outcomes). Patients stratified by ctDNA and tumor burden dynamics correspond with survival and clinical benefit. High mutation burden, high expression of immune signatures, and mutations in BRCA2 are associated with pembrolizumab molecular sensitivity, while abundant copy-number alterations and B2M loss-of-heterozygosity corresponded with resistance. Upon treatment, induction of genes expressed by T cell, B cell, and myeloid cell populations are consistent with sensitivity and resistance. We identified the upregulated expression of PLA2G2D, an immune-regulating phospholipase, as a potential biomarker of adaptive resistance to ICB. Together, these findings provide insights into the diversity of immunogenomic mechanisms that underpin pembrolizumab outcomes.
  38. Nat Commun. 2021 Aug 26. 12(1): 5147
      PTEN is frequently mutated in human cancers and PTEN mutants promote tumor progression and metastasis. PTEN mutations have been implicated in immune regulation, however, the underlying mechanism is largely unknown. Here, we report that PTENα, the isoform of PTEN, remains active in cancer bearing stop-gained PTEN mutations. Through counteraction of CD8+ T cell-mediated cytotoxicity, PTENα leads to T cell dysfunction and accelerates immune-resistant cancer progression. Clinical analysis further uncovers that PTENα-active mutations suppress host immune responses and result in poor prognosis in cancer as relative to PTENα-inactive mutations. Furthermore, germline deletion of Ptenα in mice increases cell susceptibility to immune attack through augmenting stress granule formation and limiting synthesis of peroxidases, leading to massive oxidative cell death and severe inflammatory damage. We propose that PTENα protects tumor from T cell killing and thus PTENα is a potential target in antitumor immunotherapy.
  39. Bioorg Chem. 2021 Aug 16. pii: S0045-2068(21)00648-9. [Epub ahead of print]115 105271
      In this study, a novel batch of thiazole-containing mitochondrial targeting agents were designed and synthesized. Four kinds of mitochondrial targeting moieties and six kinds of linkers were designed. Their structures were confirmed by NMR and HR-MS. The screening of antiproliferative activity revealed that most compounds displayed cytotoxicity on HeLa cancer cell. In particular, D1 has an IC50 value of 35.32 μmol·L-1 against HeLa cell. In addition, cellular respiratory activities were also tested on HeLa cancer cells. D1 had a basal oxygen consumption rate of 8.84 pmol·s-1·mL-1. Also, D1 inhibited the mitochondrial respiration of HeLa cell significantly at 5 μmol·L-1, as well as a complete inhibitory of oxygen consumption for cellular ATP coupling. Furthermore, the pKa, logP, and logD under different pH conditions of all the compounds were calculated by the ACD/Percepta-PhysChem Suite, and the results manifested the correlation between physicochemical properties and chemical activity of compounds. The results identify D1 as a promising mitochondria inhibitor and anticancer agent with appropriate physicochemical properties.
    Keywords:  Antitumor; Mitochondrial respiration; Mitochondrial targeting; Thiazole
  40. J Neuroendocrinol. 2021 Aug 09. e13027
      Changes in mitochondrial function in a variety of cells/tissues are critical for orchestrating systemic energy homeostasis and are linked to the development of obesity and many of its comorbidities. The mitochondrial translocator protein of 18 kDa (TSPO) is expressed in organs throughout the body, including the brain, liver, adipose tissue, gonads and adrenal glands, where it is implicated in regulating steroidogenesis and cellular metabolism. Prior work from our group and others has shown that, in rodents, TSPO levels are altered in adipose tissue by obesity and that modulation of TSPO activity may impact systemic glucose homeostasis. Furthermore, in vitro studies in a variety of cell types have implicated TSPO in mediating cellular energetics and substrate utilisation. Although mice with germline global TSPO deficiency (TSPO-/- ) have no reported changes in body weight under standard husbandry conditions, we hypothesised that, given the roles of TSPO in regulating mitochondrial function and cellular metabolic flexibility, these animals may have alterations in their systemic response to altered energy availability, either nutritional excess or insufficiency. In agreement with published work, compared to wild-type (TSPO+/+ ) littermates, TSPO-/- mice of both sexes did not exhibit differences in body weight on standard chow. Furthermore, following a 12-hour overnight fast, there was no difference in weight loss or compensatory food intake during re-feeding. Five weeks of feeding a high-fat diet (HFD) did not reveal any impact of the absence of TSPO on body weight gain in either male or female mice. Basal blood glucose levels and glucose clearance in a glucose tolerance test were influenced by feeding a HFD diet but not by genotype. In conclusion, in the paradigms examined, germline global deletion of TSPO did not change the physiological response to deviations in systemic energy availability at the whole organism level.
    Keywords:  TSPO; high-fat diet; metabolism; mitochondria
  41. PLoS One. 2021 ;16(8): e0244260
      Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens' tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals' behavior observation as well as by measurement of intact specimens' whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.
  42. Nutrients. 2021 Jul 28. pii: 2596. [Epub ahead of print]13(8):
      The central integration of peripheral neural signals is one mechanism by which systemic energy homeostasis is regulated. Previously, increased acute food intake following the chemical reduction of hepatic fatty acid oxidation and ATP levels was prevented by common hepatic branch vagotomy (HBV). However, possible offsite actions of the chemical compounds confound the precise role of liver energy metabolism. Herein, we used a hepatocyte PGC1a heterozygous (LPGC1a) mouse model, with associated reductions in mitochondrial fatty acid oxidation and respiratory capacity, to assess the role of liver energy metabolism in systemic energy homeostasis. LPGC1a male, but not female, mice had a 70% greater high-fat/high-sucrose (HFHS) diet-induced weight gain compared to wildtype (WT) mice (p < 0.05). The greater weight gain was associated with altered feeding behavior and lower activity energy expenditure during the HFHS diet in LPGC1a males. WT and LPGC1a mice underwent sham surgery or HBV to assess whether vagal signaling was involved in the HFHS-induced weight gain of male LPGC1a mice. HBV increased HFHS-induced weight gain (85%, p < 0.05) in male WT mice, but not LPGC1a mice. These data demonstrate a sex-specific role of reduced liver energy metabolism in acute diet-induced weight gain, and the need for a more nuanced assessment of the role of vagal signaling in short-term diet-induced weight gain.
    Keywords:  energy expenditure; energy homeostasis; food intake; liver; mitochondria; weight gain
  43. Nat Commun. 2021 08 24. 12(1): 5086
      Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.
  44. Cell. 2021 Aug 24. pii: S0092-8674(21)00945-4. [Epub ahead of print]
      Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.
    Keywords:  MSI; MSS; anti-tumor immunity; cell-cell interactions; colorectal cancer; mismatch repair-deficient; mismatch repair-proficient; scRNA-seq; spatial; tumor atlas
  45. Int J Mol Sci. 2021 Aug 10. pii: 8610. [Epub ahead of print]22(16):
      Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.
    Keywords:  cancer; dichloroacetate; myotubes; pyruvate dehydrogenase complex; pyruvate dehydrogenase kinase; skeletal muscle cells