bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒06‒07
fifty papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. EMBO Rep. 2020 Jun 04. e50287
      The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2  + IV2 , I + IV, and I + III2  + IV2 . To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1-/- fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.
    Keywords:  OXPHOS super-assembly; SCAF1/COX7A2L; metabolism; mitochondria; zebrafish
  2. Am J Physiol Heart Circ Physiol. 2020 Jun 05.
      Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that TEAD1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, though its role in cardiomyocytes is unknown. Here we show that TEAD1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss-of-function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that TEAD1 regulation of OXPHOS, in cardiomyocytes, is cell-autonomous. Taken together, our data demonstrate that TEAD1 is a crucial transcriptional node that is a cell-autonomous regulator a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis.
    Keywords:  Hippo pathway; TEAD1; heart failure; metabolism; mitochondria
  3. Cell Rep Med. 2020 May 19. 1(2): 100014
      Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc + lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc + lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies.
    Keywords:  AMPK; LKB1; Myc; OXPHOS inhibitor; biguanide; energy-sensing; lymphoma; metabolic vulnerabilities; microRNA
  4. BMC Cancer. 2020 Jun 05. 20(1): 526
      BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.
    RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us.
    CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.
    Keywords:  L-asparaginase; cancer metabolism; fatty acid oxidation; glycolysis; leukemia; mitochondrial membrane potential; mitochondrial respiration; resistance
  5. Nat Commun. 2020 Jun 01. 11(1): 2714
      Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death.
  6. Antioxid Redox Signal. 2020 May 30.
      AIMS: REDOX signaling from reactive oxygen species generated by the mitochondria (mtROS) has been implicated in cancer growth and survival. Here, we investigated the effect of AOL (5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione), a recently characterized member of the new class of mtROS suppressors (S1QELs), on human lung adenocarcinoma proteome reprogramming, bioenergetics and growth.RESULTS: AOL reduced steady-state cellular ROS levels in human lung cancer cells without altering the catalytic activity of complex I. AOL treatment induced dose-dependent inhibition of lung cancer cell proliferation and triggered a reduction in tumor growth in vivo. Molecular investigations demonstrated that AOL reprogrammed the proteome of human lung cancer cells. In particular, AOL suppressed the determinants of the Warburg effect and reduced the expression of STAT3, a master regulator of lung carcinogenesis. Comparison of the molecular changes induced by AOL and MitoTEMPO, a mtROS scavenger that is not a S1QEL, identified a core component of 217 proteins commonly altered by the two treatments, as well as drug-specific targets.
    INNOVATION: This study provides proof-of-concept data on the anticancer effect of AOL on mouse orthotopic human lung tumors. A unique dataset on proteomic reprogramming by AOL and MitoTEMPO is also provided. Last, our study revealed the repression of STAT3 by S1QEL AOL.
    CONCLUSION: Our findings demonstrate the preclinical anticancer properties of S1QEL AOL and delineate its mode of action on REDOX and cancer signaling.
  7. EMBO J. 2020 Jun 03. e103812
      It is controversial whether mitochondrial dysfunction in skeletal muscle is the cause or consequence of metabolic disorders. Herein, we demonstrate that in vivo inhibition of mitochondrial ATP synthase in muscle alters whole-body lipid homeostasis. Mice with restrained mitochondrial ATP synthase activity presented intrafiber lipid droplets, dysregulation of acyl-glycerides, and higher visceral adipose tissue deposits, poising these animals to insulin resistance. This mitochondrial energy crisis increases lactate production, prevents fatty acid β-oxidation, and forces the catabolism of branched-chain amino acids (BCAA) to provide acetyl-CoA for de novo lipid synthesis. In turn, muscle accumulation of acetyl-CoA leads to acetylation-dependent inhibition of mitochondrial respiratory complex II enhancing oxidative phosphorylation dysfunction which results in augmented ROS production. By screening 702 FDA-approved drugs, we identified edaravone as a potent mitochondrial antioxidant and enhancer. Edaravone administration restored ROS and lipid homeostasis in skeletal muscle and reinstated insulin sensitivity. Our results suggest that muscular mitochondrial perturbations are causative of metabolic disorders and that edaravone is a potential treatment for these diseases.
    Keywords:  ATP synthase; Acetyl-CoA; edaravone; insulin resistance; mitochondria
  8. Free Radic Biol Med. 2020 May 31. pii: S0891-5849(20)30421-4. [Epub ahead of print]
      Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD+) levels in mice whereas supplementing aging mice with nicotinamide riboside (NR), an NAD+ precursor, increased ovarian NAD+ content. We found that increases in ovarian NAD+ levels in aging mice led to increased number of ovarian follicles and ovulatory potential as well as increased live birth rate. NR supplementation also reduced levels of reactive oxygen species and decreased spindle anomalies in aging oocytes, together with increased mitochondrial membrane potential (ΔΨm) and decreased mitochondrial clustering. In addition, NR supplementation improved ovarian mitochondrial energy metabolism. Our data suggested that supplementation with NAD+ precursors in vivo and in vitro could be potential therapeutic approaches for treating age-related ovarian infertility.
    Keywords:  Infertility; Mitochondria; NAD(+); Ovary aging
  9. Transl Psychiatry. 2020 Jun 01. 10(1): 176
      Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.
  10. Nature. 2020 Jun;582(7810): 129-133
      Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.
  11. Int J Mol Sci. 2020 Jun 01. pii: E3977. [Epub ahead of print]21(11):
      Multimodal continuous bedside monitoring is increasingly recognized as a promising option for early treatment stratification in patients at risk for ischemia during neurocritical care. Modalities used at present are, for example, oxygen availability and subdural electrocorticography. The assessment of mitochondrial function could be an interesting complement to these modalities. For instance, flavin adenine dinucleotide (FAD) fluorescence permits direct insight into the mitochondrial redox state. Therefore, we explored the possibility of using FAD fluorometry to monitor consequences of hypoxia in brain tissue in vitro and in vivo. By combining experimental results with computational modeling, we identified the potential source responsible for the fluorescence signal and gained insight into the hypoxia-associated metabolic changes in neuronal energy metabolism. In vitro, hypoxia was characterized by a reductive shift of FAD, impairment of synaptic transmission and increasing interstitial potassium [K+]o. Computer simulations predicted FAD changes to originate from the citric acid cycle enzyme α-ketoglutarate dehydrogenase and pyruvate dehydrogenase. In vivo, the FAD signal during early hypoxia displayed a reductive shift followed by a short oxidation associated with terminal spreading depolarization. In silico, initial tissue hypoxia followed by a transient re-oxygenation phase due to glucose depletion might explain FAD dynamics in vivo. Our work suggests that FAD fluorescence could be readily used to monitor mitochondrial function during hypoxia and represents a potential diagnostic tool to differentiate underlying metabolic processes for complementation of multimodal brain monitoring.
    Keywords:  FAD; brain; computational modeling; hypoxia; mitochondria; spreading depolarization
  12. Mol Metab. 2020 May 29. pii: S2212-8778(20)30101-0. [Epub ahead of print] 101027
      OBJECTIVES: Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and in programmed cell death. The relevant role of this protein is underlined by the fact that mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, the present study sought to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction.METHODS: We developed a novel AIF-deficient mouse strain and assessed, by molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we carried out ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown.
    RESULTS: AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming towards anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose assimilation transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Interestingly, about 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism towards anaerobic glycolysis. Heterozygous AIF-/+ females displayed a progressive bone marrow, thymus, and spleen cellular loss. In addition, about 10% of AIF-/+ females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died around 5 weeks of age. AIF-/+ with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype seemed triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF-/+ females by supplying dams and newborns with an antioxidant in drinking water.
    CONCLUSION: In a single knockout model and at three different levels (cell, embryo, and adult mice) we demonstrated that, by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by shedding new light on the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies.
    Keywords:  AIF; Caspase-independent cell death; Hydrocephaly; Metabolism; Mitochondria; OXPHOS
  13. Cancers (Basel). 2020 May 31. pii: E1427. [Epub ahead of print]12(6):
      Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.
    Keywords:  RL2; TOM70; breast cancer; cell death; lactaptin; mitochondria; κ-Casein
  14. Theranostics. 2020 ;10(13): 5879-5894
      Rationale: Interleukin 22 (IL-22) is an epithelial survival cytokine that is at present being explored as therapeutic agents for acute and chronic liver injury. However, its molecular basis of protective activities remains poorly understood. Methods: Here we demonstrate that IL-22 inhibits the deteriorating metabolic states induced by stimuli in hepatocytes. Utilizing cell biological, molecular, and biochemical approaches, we provide evidence that IL-22 promotes oxidative phosphorylation (OXPHOS) and glycolysis and regulates the metabolic reprogramming related transcriptional responses. Results: IL-22 controls metabolic regulators and enzymes activity through the induction of AMP-activated protein kinase (AMPK), AKT and mammalian target of rapamycin (mTOR), thereby ameliorating mitochondrial dysfunction. The upstream effector lncRNA H19 also participates in the controlling of these metabolic processes in hepatocytes. Importantly, amelioration of liver injury by IL-22 through activation of metabolism relevant signaling and regulation of mitochondrial function are further demonstrated in cisplatin-induced liver injury and steatohepatitis. Conclusions: Collectively, our results reveal a novel mechanism underscoring the regulation of metabolic profiles of hepatocytes by IL-22 during liver injury, which might provide useful insights from the bench to the clinic in treating and preventing liver diseases.
    Keywords:  glycolysis; lncRNA H19; mitochondria; oxidative phosphorylation; reactive oxygen species
  15. Cancer Sci. 2020 Jun 03.
      Neuroblastoma (NB) is a childhood malignancy originating from the sympathetic nervous system, which accounts for approximately 15% of all pediatric cancer-related deaths. As the 5-year survival rate of patients with high-risk NB is < 50%, novel therapeutic strategies for NB patients are urgently required. Nonaethylene glycol mono ('4-iodo-4-biphenyl) ester (9bw) is a polyethylene glycol derivative, synthesized by modifying a compound originally extracted from filamentous bacteria. Although 9bw shows remarkable inhibition of tumor cell growth, the underlying mechanisms remain unclear. Here, we examined the efficacy of 9bw on human NB-derived cells, and investigated the molecular mechanisms underlying the cytotoxic effects of 9bw on these cells. Our results indicated that 9bw induced cell death in NB cells by decreasing the production of ATP. Metabolome analysis and measurement of oxygen consumption indicated that 9bw markedly suppressed oxidative phosphorylation (OXPHOS). Further analyses indicated that 9bw inhibited the activity of mitochondrial respiratory complex I. Moreover, we showed that 9bw inhibited growth of NB in vivo. Based on the results of the present study, 9bw is a good candidate as a novel agent for treatment of NB.
    Keywords:  anticancer drug; mitochondrial respiratory complex I; neuroblastoma; oxidative phosphorylation; polyethylene glycol derivative
  16. Biochemistry. 2020 Jun 03.
      Cancer cells are often characterized by elevated levels of mitochondria-bound hexokinase II (HKII), which facilitates their survival, proliferation and metastasis. Here, we have designed a cancer-selective cell-penetrating peptide (CPP) by covalently coupling a short penetration accelerating sequence (PAS) to the mitochondrial membrane-binding N-terminal 15 amino acids of HKII (pHK). PAS-pHK mediates efficient cellular uptake and cytosolic delivery of a synthetic mimic of miR-126, a tumor suppressor miRNA downregulated in many malignancies. Following uptake by breast cancer MCF-7 cells, the CPP-miRNA conjugate is distributed throughout the cytosol and shows strong colocalization with mitochondria, where PAS-pHK induces depolarization of mitochondrial membrane potential, inhibition of metabolic activities, depletion of intracellular ATP levels, release of cytochrome c and, finally, apoptosis. Concomitantly, the miR-126 cargo synergistically enhances the anticancer effects of PAS-pHK. Importantly, PAS-pHK-miR-126 is not toxic to noncancerous MCF-10A and HEK-93 cells. Our results demonstrate the potential of PAS-pHK mediated delivery of miRNA mimics as a novel cancer-selective therapeutic strategy.
  17. Mol Cancer Res. 2020 Jun 03. pii: molcanres.0364.2020. [Epub ahead of print]
      Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion and metastasis but how stromal lipids influence these processes remain to be defined. Here we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment. Implications: This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content and inflammatory response.
  18. Mol Cell Biochem. 2020 Jun 05.
      NME4, also designated nm23-H4 or NDPK-D, has been known for years for its well-established roles in the synthesis of nucleoside triphosphates, though; little has been known regarding the differential metabolites involved as well as the biological roles NME4 plays in proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells. To understand the biological roles of NME4 in ESCC cells, lentiviral-based short hairpin RNA interference (shRNA) vectors were constructed and used to stably knock down NME4. Then, the proliferative and invasive variations were assessed using MTT, Colony formation and Transwell assays. To understand the metabolites involved after silencing of NME4 in ESCC cells, widely targeted metabolomic screening was taken. It was discovered that silencing of NME4 can profoundly suppress the proliferation and invasion in ESCC cells in vitro. Metabolically, a total of 11 differential metabolites were screened. KEGG analyses revealed that Tryptophan, Riboflavin, Purine, Nicotinate, lysine degradation, and Linoleic acid metabolism were also involved in addition to the well-established nucleotides metabolism. Some of these differential metabolites, say, 2-Picolinic Acid, Nicotinic Acid and Pipecolinic Acid were suggested to be associated with tumor immunomodulation. The data we described here support the idea that metabolisms occurred in mitochondrial was closely related to tumor immunity.
    Keywords:  ESCC; Invasion; Metabolism; NME4; Widely targeted metabolomics
  19. Antioxidants (Basel). 2020 Jun 02. pii: E472. [Epub ahead of print]9(6):
      Mitochondria are quantifiably the most important sources of superoxide (O2●-) and hydrogen peroxide (H2O2) in mammalian cells. The overproduction of these molecules has been studied mostly in the contexts of the pathogenesis of human diseases and aging. However, controlled bursts in mitochondrial ROS production, most notably H2O2, also plays a vital role in the transmission of cellular information. Striking a balance between utilizing H2O2 in second messaging whilst avoiding its deleterious effects requires the use of sophisticated feedback control and H2O2 degrading mechanisms. Mitochondria are enriched with H2O2 degrading enzymes to desensitize redox signals. These organelles also use a series of negative feedback loops, such as proton leaks or protein S-glutathionylation, to inhibit H2O2 production. Understanding how mitochondria produce ROS is also important for comprehending how these organelles use H2O2 in eustress signaling. Indeed, twelve different enzymes associated with nutrient metabolism and oxidative phosphorylation (OXPHOS) can serve as important ROS sources. This includes several flavoproteins and respiratory complexes I-III. Progress in understanding how mitochondria generate H2O2 for signaling must also account for critical physiological factors that strongly influence ROS production, such as sex differences and genetic variances in genes encoding antioxidants and proteins involved in mitochondrial bioenergetics. In the present review, I provide an updated view on how mitochondria budget cellular H2O2 production. These discussions will focus on the potential addition of two acyl-CoA dehydrogenases to the list of ROS generators and the impact of important phenotypic and physiological factors such as tissue type, mouse strain, and sex on production by these individual sites.
    Keywords:  bioenergetics; hydrogen peroxide; isopotential groups; mitochondria; reactive oxygen species; sex differences; substrate preferences
  20. Front Physiol. 2020 ;11 491
      Background: We recently reported a method using positron emission tomography (PET) and the tracer 18F-labeled tetraphenylphosphonium (18F-TPP+) for mapping the tissue (i.e., cellular plus mitochondrial) membrane potential (ΔΨT) in the myocardium. The purpose of this work is to provide additional experimental evidence that our methods can be used to observe transient changes in the volume of distribution for 18F-TPP+ and mitochondrial membrane potential (ΔΨm).Methods: We tested these hypotheses by measuring decreases of 18F-TPP+ concentration elicited when a proton gradient uncoupler, BAM15, is administered by intracoronary infusion during PET scanning. BAM15 is the first proton gradient uncoupler shown to affect the mitochondrial membrane without affecting the cellular membrane potential. Preliminary dose response experiments were conducted in two pigs to determine the concentration of BAM15 infusate necessary to perturb the 18F-TPP+ concentration. More definitive experiments were performed in two additional pigs, in which we administered an intravenous bolus plus infusion of 18F-TPP+ to reach secular equilibrium followed by an intracoronary infusion of BAM15.
    Results: Intracoronary BAM15 infusion led to a clear decrease in 18F-TPP+ concentration, falling to a lower level, and then recovering. A second BAM15 infusion reduced the 18F-TPP+ level in a similar fashion. We observed a maximum depolarization of 10 mV as a result of the BAM15 infusion.
    Summary: This work provides evidence that the total membrane potential measured with 18F-TPP+ PET is sensitive to temporal changes in mitochondrial membrane potential.
    Keywords:  BAM15; mitochondrial membrane potential; positron emission tomography; tetraphenylphosphonium; tissue membrane potential
  21. J Exp Med. 2020 Aug 03. pii: e20191920. [Epub ahead of print]217(8):
      CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.
  22. Biochim Biophys Acta Mol Basis Dis. 2020 May 29. pii: S0925-4439(20)30198-8. [Epub ahead of print] 165851
      ERK and Akt have been shown to regulate cell sensitivity to death-inducing stress by phosphorylating GSK-3β, a major modulator of the threshold for mitochondrial permeability transition. Here we examined intra-mitochondrial localization of the pro-survival kinases and their regulation by phosphatases. Stepwise trypsin digestion of mitochondria isolated from HEK293 or H9c2 cells was performed, and immunoblotting revealed that GSK-3β and ERK localized dominantly in the outer membrane (OM), while Akt resided at comparable levels in OM, the inner membrane (IM) and the matrix. Treatment with IGF-1 increased the protein level of Akt in the matrix, while ERK and GSK-3β protein levels were increased in OM. Simultaneously, IGF-1 treatment elevated the level of Thr202/Tyr204-phospho-ERK in IM and matrix and levels of Ser473-phospho-Akt and Ser9-phospho-GSK-3β in OM, IM and matrix. Exposing cells to reactive oxygen species (ROS) by using antimycin A increased the levels of DUSP5 and PHLPP-1 mainly in OM and induced dephosphorylation of Akt, ERK and GSK-3β. The mitochondrial localization of DUSP5 was confirmed by experiments with mitochondria purified by Percoll gradient centrifugation and by transfection of cells with GFP-tagged DUSP5. Knockdown of either DUSP5 or PHLPP-1 increased the levels of both Thr202/Tyr204-phospho-ERK and Ser473-phospho-Akt in mitochondria. Cell death induced by antimycin A was suppressed by siRNA-mediated knockdown of DUSP5. The results suggest that Akt and ERK in mitochondria show distinct intra-mitochondrial localization and crosstalk in GSK-3β regulation and that recruitment of DUSP5 as well as PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling.
    Keywords:  Akt; DUSP5; ERK; GSK-3β; Mitochondria; PHLPP-1
  23. Biochim Biophys Acta Mol Basis Dis. 2020 May 27. pii: S0925-4439(20)30193-9. [Epub ahead of print] 165846
      Malignant conversion of cancer cells requires efficient mitochondria reprogramming orchestrated by hundreds of genes. The transformation includes increased energy demand, biosynthesis of precursors, and reactive oxygen species needed to accelerate cell growth, proliferation, and survival. The reprogramming involves complex gene alterations that have not been methodically curated. Therefore, we systematically analyzed the literature of cancer-related genes in mitochondria. Through the analysis of >2500 PubMed abstracts and >1600 human genes, we identified 228 genes showing clear roles in cancer. Each gene was classified according to their homeostatic function, together with the pathological transitions that contribute to specific cancer hallmarks. The potential clinical relevance of these hallmarks and genes is discussed by representative examples and validated by detecting differences in gene expression levels across 16 different types of cancer. A compendium, including the gene functions and alterations underpinning cancer progression, can be explored at
    Keywords:  Cancer; Hallmarks of cancer; Mitochondria; Mitochondrial proteins
  24. Redox Biol. 2020 Apr 20. pii: S2213-2317(20)30022-7. [Epub ahead of print] 101465
      Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail. Degradation of damaged mitochondria is a critical step of MQC, and here we have used hPSC-derived RGCs (hRGCs) to analyze how altered mitochondrial degradation pathways in hRGCs affect their survival. Using pharmacological methods, we have investigated the role of the proteasomal and endo-lysosomal pathways in degrading damaged mitochondria in hRGCs and their precursor stem cells. We found that upon mitochondrial damage induced by the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), hRGCs more efficiently degraded mitochondria than did their precursor stem cells. We further identified that for degrading damaged mitochondria, stem cells predominantly use the ubiquitine-proteasome system (UPS) while hRGCs use the endo-lysosomal pathway. UPS inhibition causes apoptosis and cell death in stem cells, while hRGC viability is dependent on the endo-lysosomal pathway but not on the UPS pathway. These findings suggest that manipulation of the endo-lysosomal pathway could be therapeutically relevant for RGC protection in treating optic neuropathies associated with mitophagy defects. Endo-lysosome dependent cell survival is also conserved in other human neurons as we found that differentiated human cerebral cortical neurons also degenerated upon endo-lysosomal inhibition but not with proteasome inhibition.
    Keywords:  Autophagy; Glaucoma; Human retinal ganglion cells (hRGCs); Mitophagy; Stem cells; Ubiquitine-proteasome system (UPS)
  25. Neuroscience. 2020 Jun 01. pii: S0306-4522(20)30342-0. [Epub ahead of print]
      NAD(P)+ transhydrogenase (NNT) links redox states of the mitochondrial NAD(H) and NADP(H) via a reaction coupled to proton-motive force across the inner mitochondrial membrane. NNT is believed to be ubiquitously present in mammalian cells, but its expression may vary substantially in different tissues. The present study investigated the tissue distribution and possible roles of NNT in the mouse brain. The pons exhibited high NNT expression/activity, and immunohistochemistry revealed intense NNT labeling in neurons from brainstem nuclei. In some of these regions, neuronal NNT labeling was strongly colocalized with enzymes involved in the biosynthesis of 5-hydroxytryptamine (5-HT) and nitric oxide (•NO), which directly or indirectly require NADPH. Behavioral tests were performed in mice lacking NNT activity (Nnt-/-, mice carrying the mutated NntC57BL/6J allele from the C57BL/6J strain) and the Nnt+/+ controls. Our data demonstrated that aged Nnt-/- mice (18-20 months old), but not adult mice (3-4 months old), showed an increased immobility time in the tail suspension test that was reversed by fluoxetine treatment, providing evidence of depressive-like behavior in these mice. Aged Nnt-/- mice also exhibited behavioral changes and impaired locomotor activity in the open field and rotarod tests. Despite the colocalization between NNT and •NO synthase, the S-nitrosation and cGMP levels were independent of the Nnt genotype. Taken together, our results indicated that NNT is unevenly distributed throughout the brain and associated with 5-THergic and •NOergic neurons. The lack of NNT led to alterations in brain functions related to mood and motor behavior/performance in aged mice.
    Keywords:  C57BL/6; NADPH; mitochondria; monoamine; nicotinamide nucleotide transhydrogenase; redox balance
  26. Nat Rev Endocrinol. 2020 Jun 03.
      Perturbations in metabolic processes are associated with diseases such as obesity, type 2 diabetes mellitus, certain infections and some cancers. A resurgence of interest in creatine biology is developing, with new insights into a diverse set of regulatory functions for creatine. This resurgence is primarily driven by technological advances in genetic engineering and metabolism as well as by the realization that this metabolite has key roles in cells beyond the muscle and brain. Herein, we highlight the latest advances in creatine biology in tissues and cell types that have historically received little attention in the field. In adipose tissue, creatine controls thermogenic respiration and loss of this metabolite impairs whole-body energy expenditure, leading to obesity. We also cover the various roles that creatine metabolism has in cancer cell survival and the function of the immune system. Renewed interest in this area has begun to showcase the therapeutic potential that lies in understanding how changes in creatine metabolism lead to metabolic disease.
  27. Cancer Discov. 2020 Jun 04. pii: CD-20-0329. [Epub ahead of print]
      Older melanoma patients (>50 years old) have poorer prognoses and response rates to targeted therapy compared to young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or co-cultured with aged fibroblasts, they increase the uptake of lipids, via the fatty acid transporter, fatty acid transport protein (FATP) 2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids, and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals.
  28. Int Rev Cell Mol Biol. 2020 ;pii: S1937-6448(20)30010-1. [Epub ahead of print]354 1-61
      Initially discovered as a protease responsible for degradation of misfolded or damaged proteins, the mitochondrial Lon protease (Lonp1) turned out to be a multifaceted enzyme, that displays at least three different functions (proteolysis, chaperone activity, binding of mtDNA) and that finely regulates several cellular processes, within and without mitochondria. Indeed, LONP1 in humans is ubiquitously expressed, and is involved in regulation of response to oxidative stress and, heat shock, in the maintenance of mtDNA, in the regulation of mitophagy. Furthermore, its proteolytic activity can regulate several biochemical pathways occurring totally or partially within mitochondria, such as TCA cycle, oxidative phosphorylation, steroid and heme biosynthesis and glutamine production. Because of these multiple activities, Lon protease is highly conserved throughout evolution, and mutations occurring in its gene determines severe diseases in humans, including a rare syndrome characterized by Cerebral, Ocular, Dental, Auricular and Skeletal anomalies (CODAS). Finally, alterations of LONP1 regulation in humans can favor tumor progression and aggressiveness, further highlighting the crucial role of this enzyme in mitochondrial and cellular homeostasis.
    Keywords:  CODAS; Colon cancer; LONP1; Lon protease; Mitochondria; Proteotoxic stress; mtDNA
  29. Chem Res Toxicol. 2020 Jun 03.
      Mitochondria have a plethora of functions within a eukaryotic cell, ranging from energy production, cell signaling and protein cofactor synthesis to various aspects of metabolism. Mitochondrial dysfunction is known to cause over 200 named disorders and has been implicated in many human diseases and aging. Mitochondria have their own genetic material, mitochondrial DNA (mtDNA), which encodes 13 protein subunits in the oxidative phosphorylation system and a full set of transfer and ribosomal RNAs. Although more than 99% of the proteins in mitochondria are nuclear DNA (nDNA)-encoded, the integrity of mtDNA is critical for mitochondrial functions, as evidenced by mitochondrial diseases sourced from mtDNA mutations and depletions and the vital role of fragmented mtDNA molecules in cell signaling pathways. Previous research has shown that mtDNA is an important target of genotoxic assaults by a variety of chemical and physical factors. This Perspective discusses the prevalence of mtDNA damage by comparing the abundance of lesions in mDNA and nDNA, and summarizes current knowledge on the biological pathways to cope with mtDNA damage, including mtDNA repair, mtDNA degradation, and mitochondrial fission and fusion. Also, emerging roles of mtDNA damage in mutagenesis and immune responses are reviewed.
  30. Cancers (Basel). 2020 Jun 01. pii: E1440. [Epub ahead of print]12(6):
      Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and will become the second most common cause of cancer-associated mortality by 2030. The poor prognosis arises from a lack of sensitive biomarkers, limited therapeutic options, and the astonishingly high recurrence rate after surgery of 60-80%. The factors driving this recurrence, however, remain enigmatic. Therefore, we generated patient-derived organoids (PDOs) from early- and late-recurrent PDAC patients. Cellular identity of PDOs was confirmed by qPCR, ddPCR, and IHC analyses. This is the first study investigating the metabolism in PDOs of different, clinically significant PDAC entities by untargeted GC/MS profiling. Partial least square discriminant analysis unveiled global alterations between the two sample groups. We identified nine metabolites to be increased in early recurrent PDOs in comparison to late recurrent PDOs. More than four-times increased were fumarate, malate, glutamate, aspartate, and glutamine. Hence, α-keto acids were elevated in PDO-conditioned medium derived from early recurrent patients. We therefore speculate that an increased anaplerotic metabolism fuels the Krebs-cycle and a corresponding higher accessibility to energy fastens the recurrence in PDAC patients. Therein, a therapeutic intervention could delay PDAC recurrence and prolong survival of affected patients or could serve as biomarker to predict recurrence in the future.
    Keywords:  GC/MS; PDAC; PDO culture; anaplerotic TCA-cycle; glutamate; metabolomics; pancreatic ductal adenocarcinoma; patient derived organoids; recurrence; relapse
  31. Cell Biochem Biophys. 2020 Jun 01.
      It has been proposed that a mitochondrial switch involving a high mitochondrial superoxide production is associated with cancer metastasis. We here report an EPR analysis of ROS production using cyclic hydroxylamines in superinvasive SiHa-F3 compared with less invasive SiHa wild-type human cervix cancer cells. Using the CMH probe, no significant difference was observed in the overall level of ROS between SiHa and SiHa-F3 cells. However, using mitochondria-targeted cyclic hydroxylamine probe mitoTEMPO-H, we detected a significantly higher mitochondrial ROS content in SiHa-F3 compared with the wild-type SiHa cells. To investigate the nature of mitochondrial ROS, we overexpressed superoxide dismutase 2, a SOD isoform exclusively localized in mitochondria, in SiHa-F3 superinvasive cells. A significantly lower signal was detected in SiHa-F3 cells overexpressing SOD2 compared with SiHa-F3. Despite some limitations discussed in the paper, our EPR results suggest that mitochondrial ROS (at least partly superoxide) are produced to a larger extent in superinvasive cancer cells compared with less invasive wild-type cancer cells.
  32. Nat Commun. 2020 Jun 03. 11(1): 2790
      Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD+ (kynurenine) pathway gene, reduces LD accumulation during aging and extends lifespan. Mechanistically, this LD accumulation during aging is not linked to NAD+ levels, but is anti-correlated with metabolites of the shikimate and aromatic amino acid biosynthesis (SA) pathways (upstream of BNA2), which produce tryptophan (the Bna2p substrate). We provide evidence that over-expressed BNA2 skews glycolytic flux from LDs towards the SA-BNA pathways, effectively reducing LDs. Importantly, we find that accumulation of LDs does not shorten lifespan, but does protect aged cells against stress. Our findings reveal how lipid accumulation impacts longevity, and how aging cell metabolism can be rewired to modulate lipid accumulation independently from longevity.
  33. Breast Cancer Res. 2020 Jun 05. 22(1): 60
      BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC).METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer.
    RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer.
    CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.
    Keywords:  Akt; ERK; Fluorescence microscopy; Metastasis; Mitochondrial fission; Mitochondrial fusion; Triple-negative breast cancer
  34. Nat Metab. 2020 May;2(5): 413-431
      Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo. Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.
  35. Molecules. 2020 May 29. pii: E2545. [Epub ahead of print]25(11):
      Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2-• that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.
    Keywords:  N-alkylaminoferrocene; cancer; mitochondrion; prodrug; reactive oxygen species
  36. Int J Pharm. 2020 Jun 01. pii: S0378-5173(20)30449-X. [Epub ahead of print] 119465
      Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and thisdepends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.
    Keywords:  Ataxia-Telangiectasia Mutated Kinase; Triphenylphosphonium-Functionalized hyperbranched polymer; breast adenocarcinoma; chloroquine; mitochondria
  37. Toxicol In Vitro. 2020 Jun 02. pii: S0887-2333(20)30457-4. [Epub ahead of print] 104907
      Oligomycin is a classical mitochondrial reagent that binds to the proton channel on the Fo component of ATP synthase. As a result, oligomycin blocks mitochondrial ATP synthesis, proton translocation, and O2 uptake. Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O2 uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line. Proton fluxes plateau at oligomycin concentrations in the region 0.25-5 μM. At the plateau, fluxes are lower than expected for the classical mitochondrial permeability transition pore, although in H1229 cells, fluxes increase to levels consistent with pore opening at higher oligomycin concentrations. Uncoupling is observed in cells metabolizing either pyruvate or lactate and reversed by addition of glucose to restore ATP synthesis. Uncoupling is not sensitive to cyclosporin A and is not reversed by the ANT inhibitor bongkrekic acid. However, bongkrekic acid inhibits uncoupling if added before oligomycin, which we interpret in terms of maintenance of mitochondrial ATP levels.
    Keywords:  Bongkrekic acid; Bz-423; HepG2; Mitochondrial permeability transition pore; Oligomycin; Proton uncoupling
  38. J Cancer. 2020 ;11(15): 4397-4405
      Although the roles and underlying mechanisms of other PDK family members (i.e., PDK1, PDK2 and PDK3) in tumor progression have been extensively investigated and are well understood, the functions and underlying molecular mechanisms of pyruvate dehydrogenase kinase 4 (PDK4) in the tumorigenesis and progression of various cancers [including hepatocellular carcinoma (HCC)] remain largely unknown. In this study, we examined the expression profile of PDK4 in HCC clinical tissue specimens and the roles of PDK4 in the proliferation, tumorigenicity, motility and invasion of HCC cells. The immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) results revealed that PDK4 was significantly downregulated in the cohort of HCC clinical specimens. Additionally, PDK4 protein was found in both the nucleus and cytoplasm of HCC cells based on an immunofluorescence (ICC) assay, and PDK4 protein was also found in the nucleus and cytoplasm of cancer cells contained in HCC clinical specimens based on IHC. The CCK-8 assay and cell colony formation assay demonstrated that stable depletion of endogenous PDK4 by lentivirus-mediated RNA interference (RNAi) markedly promoted the proliferation of HCC cell lines (i.e., BEL-7402 and BEL-7404 cells) in vitro, while PDK4 silencing significantly enhanced the tumorigenic ability of BEL-7404 cells in vivo. In addition to enhance proliferation and tumorigenesis induced by PDK4 silencing, additional studies demonstrated that knockdown of PDK4 led to increase migration and invasion of BEL-7402 and BEL-7404 cells in vitro. Taken together, these findings suggest that the loss of PDK4 expression contributes to HCC malignant progression.
    Keywords:  Cell proliferation; Hepatocellular carcinoma; Invasion; Migration; Pyruvate dehydrogenase kinase 4
  39. Cancer Discov. 2020 Jun;10(6): 768-770
      Tumor cells maintain a reverse pH gradient relative to normal cells, conferring cell-intrinsic and cell-extrinsic benefits that sustain tumor growth. In this issue of Cancer Discovery, Galenkamp and colleagues reveal that NHE7 mediates acidification of the trans-Golgi network in pancreatic ductal adenocarcinoma, which is critical for the maintenance of cytosolic pH and consequently tumor growth.See related article by Galenkamp et al., p. 822.
  40. Talanta. 2020 Sep 01. pii: S0039-9140(20)30291-5. [Epub ahead of print]217 121000
      Hydrogen peroxide (H2O2), an important member of the family of reactive oxygen species (ROS), has a significant impact on cell signal transduction, energy conversion and immune responses of living organisms. Therefore, accurate detection of the content of H2O2 in living cells is of vital importance. In this paper, we report on the synthesis of a novel colorimetric and near-infrared fluorescent probe HAA, a heterocyclic aromatic amine with acetyl group for the specific detection of both exogenous and endogenous H2O2 in living cells. Our results show that the probe not only possesses high specificity and sensitivity, but also has advantages of low cytotoxicity and good biocompatibility. Theoretical computations elucidated the luminescence and quenching mechanism of HAA in the absence and presence of H2O2. In addition, HAA was applied to the determination of H2O2 in human serum and the imaging of endogenous H2O2 in living cells, during which it demonstrated excellent performance and good potential for future bioanalysis applications.
    Keywords:  Colorimetric; Fluorescent probe; Hydrogen peroxide; Live cell imaging; Near-infrared
  41. Cell Metab. 2020 Jun 02. pii: S1550-4131(20)30252-7. [Epub ahead of print]31(6): 1041-1043
      In this issue of Cell Metabolism, Pirinen et al. (2020) show that disruption in NAD+ homeostasis is a key component of the pathogenesis of mitochondrial myopathy in humans that can be targeted by the administration of the NAD+ precursor niacin, identifying NAD+ boosting as a potential treatment for this devastating disease.
  42. Circulation. 2020 Jun 01.
      Background: Recent evidence suggests that cancer and cardiovascular diseases are associated. Chemotherapy drugs are known to result in cardiotoxicity, and studies have shown that heart failure (HF) and stress correlate with poor cancer prognosis. Yet, whether cardiac remodeling in the absence of HF is sufficient to promote cancer is unknown. Methods: To investigate the effect of early cardiac remodeling on tumor growth and metastasis colonization, we used transverse aortic constriction (TAC), a model for pressure overload-induced cardiac hypertrophy, and followed it by cancer cell implantation. Results: TAC-operated mice developed larger primary tumors with higher proliferation rate and displayed more metastatic lesions compared with controls. Serum derived from TAC-operated mice potentiated cancer cell proliferation in vitro, suggesting the existence of secreted tumor-promoting factors. Using RNA-seq data, we identified elevated mRNA levels of Periostin in the hearts of TAC-operated mice. Periostin levels were also found high in the serum following TAC. Interestingly, depletion of Periostin from the serum abrogated the proliferation of cancer cells, conversely, the addition of Periostin enhanced cancer cell proliferation in vitro. Collectively, this is the first study to show that early cardiac remodeling nurtures tumor growth and metastasis, and therefore promotes cancer progression. Conclusions: Our study highlights the importance of early diagnosis and treatment of cardiac remodeling as it may attenuate cancer progression and improve cancer outcome.
    Keywords:  Periostin; cancer and stroke; transverse aortic constriction
  43. Cell Rep. 2020 Jun 02. pii: S2211-1247(20)30671-9. [Epub ahead of print]31(9): 107701
      The mechanistic contributions of cancer-associated fibroblasts (CAFs) in breast cancer progression remain to be fully understood. While altered glucose metabolism in CAFs could fuel cancer cells, how such metabolic reprogramming emerges and is sustained needs further investigation. Studying fibroblasts isolated from patients with benign breast tissues and breast cancer, in conjunction with multiple animal models, we demonstrate that CAFs exhibit a metabolic shift toward lactate and pyruvate production and fuel biosynthetic pathways of cancer cells. The depletion or suppression of the lactate production of CAFs alter the tumor metabolic profile and impede tumor growth. The glycolytic phenotype of the CAFs is in part sustained through epigenetic reprogramming of HIF-1α and glycolytic enzymes. Hypoxia induces epigenetic reprogramming of normal fibroblasts, resulting in a pro-glycolytic, CAF-like transcriptome. Our findings suggest that the glucose metabolism of CAFs evolves during tumor progression, and their breast cancer-promoting phenotype is partly mediated by oxygen-dependent epigenetic modifications.
    Keywords:  breast cancer; cancer-associated fibroblasts; epigenetic alterations; hypoxia; metabolism
  44. Life Sci Alliance. 2020 Jul;pii: e201900620. [Epub ahead of print]3(7):
      Recent breakthroughs in live-cell imaging have enabled visualization of cristae, making it feasible to investigate the structure-function relationship of cristae in real time. However, quantifying live-cell images of cristae in an unbiased way remains challenging. Here, we present a novel, semi-automated approach to quantify cristae, using the machine-learning Trainable Weka Segmentation tool. Compared with standard techniques, our approach not only avoids the bias associated with manual thresholding but more efficiently segments cristae from Airyscan and structured illumination microscopy images. Using a cardiolipin-deficient cell line, as well as FCCP, we show that our approach is sufficiently sensitive to detect perturbations in cristae density, size, and shape. This approach, moreover, reveals that cristae are not uniformly distributed within the mitochondrion, and sites of mitochondrial fission are localized to areas of decreased cristae density. After a fusion event, individual cristae from the two mitochondria, at the site of fusion, merge into one object with distinct architectural values. Overall, our study shows that machine learning represents a compelling new strategy for quantifying cristae in living cells.
  45. EMBO Rep. 2020 Jun 02. e48686
      Impairment of PINK1/parkin-mediated mitophagy is currently proposed to be the molecular basis of mitochondrial abnormality in Parkinson's disease (PD). We here demonstrate that PINK1 directly phosphorylates Drp1 on S616. Drp1S616 phosphorylation is significantly reduced in cells and mouse tissues deficient for PINK1, but unaffected by parkin inactivation. PINK1-mediated mitochondrial fission is Drp1S616 phosphorylation dependent. Overexpression of either wild-type Drp1 or of the phosphomimetic mutant Drp1S616D , but not a dephosphorylation-mimic mutant Drp1S616A , rescues PINK1 deficiency-associated phenotypes in Drosophila. Moreover, Drp1 restores PINK1-dependent mitochondrial fission in ATG5-null cells and ATG7-null Drosophila. Reduced Drp1S616 phosphorylation is detected in fibroblasts derived from 4 PD patients harboring PINK1 mutations and in 4 out of 7 sporadic PD cases. Taken together, we have identified Drp1 as a substrate of PINK1 and a novel mechanism how PINK1 regulates mitochondrial fission independent of parkin and autophagy. Our results further link impaired PINK1-mediated Drp1S616 phosphorylation with the pathogenesis of both familial and sporadic PD.
    Keywords:  Parkinson’s disease; autophagy; human dermal fibroblasts; mitochondrial dynamics; parkin
  46. Mol Ther Methods Clin Dev. 2020 Jun 12. 17 1071-1078
      Leigh syndrome, or infantile necrotizing subacute encephalopathy (OMIM #256000), is one of the most common manifestations of mitochondrial dysfunction, due to mutations in more than 75 genes, with mutations in respiratory complex I subunits being the most common cause. In the present study, we used the recently described PHP.B serotype, characterized by efficient capacity to cross the blood-brain barrier, to express the hNDUFS4 gene in the Ndufs4 -/- mouse model of Leigh disease. A single intravenous injection of PHP.B-hNDUFS4 in adult Ndufs4 -/- mice led to a normalization of the body weight, marked amelioration of the rotarod performance, delayed onset of neurodegeneration, and prolongation of the lifespan up to 1 year of age. hNDUFS4 protein was expressed in virtually all brain regions, leading to a partial recovery of complex I activity. Our findings strongly support the feasibility and effectiveness of adeno-associated viral vector (AAV)-mediated gene therapy for mitochondrial disease, particularly with new serotypes showing increased permeability to the blood-brain barrier in order to achieve widespread expression in the central nervous system.
    Keywords:  AAV; Leigh syndrome; Ndufs4; OXPHOS; PHP.B; complex I; gene therapy; mitochondrial diseases
  47. Nature. 2020 Jun 03.
      Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.
  48. Oncogene. 2020 Jun 03.
      Endophilin B1, also known as BAX-interacting protein 1 (BIF-1), is part of the endophilin B protein family, and is a multifunctional protein involved in the regulation of apoptosis, autophagy, and mitochondrial morphology. The role of BIF-1 in cancer is controversial since previous reports indicated to both tumor-promoting and tumor-suppressive roles, perhaps depending on the cancer cell type. In the present study, we report that BIF-1 is significantly downregulated in both primary and metastatic melanomas, and that patients with high levels of BIF-1 expression exhibited a better overall survival. Depleting BIF-1 using CRISPR/Cas9 technology in melanoma cells resulted in higher proliferation rates both in vitro and in vivo, a finding that was associated with increased ATP production, metabolic acidification, and mitochondrial respiration. We also observed mitochondrial hyperpolarization, but no increase in the mitochondrial content of BIF-1-knockout melanoma cells. In contrast, such knockout melanoma cells were equally sensitive to anticancer drug- or UV irradiation-induced cell death, and exhibited similar autophagic activities as compared with control cells. Taken together, it appears that downregulation of BIF-1 contributes to tumorigenesis in cutaneous melanoma by upregulating mitochondrial respiration and metabolism, independent of its effect on apoptosis and autophagy.
  49. Int Rev Cell Mol Biol. 2020 ;pii: S1937-6448(20)30005-8. [Epub ahead of print]354 107-164
      Cisplatin is one of the most potent and widely used chemotherapeutic agent in the treatment of several solid tumors, despite the high toxicity and the frequent relapse of patients due to the onset of drug resistance. Resistance to chemotherapeutic agents, either intrinsic or acquired, is currently one of the major problems in oncology. Thus, understanding the biology of chemoresistance is fundamental in order to overcome this challenge and to improve the survival rate of patients. Studies over the last 30 decades have underlined how resistance is a multifactorial phenomenon not yet completely understood. Recently, tumor metabolism has gained a lot of interest in the context of chemoresistance; accumulating evidence suggests that the rearrangements of the principal metabolic pathways within cells, contributes to the sensitivity of tumor to the drug treatment. In this review, the principal metabolic alterations associated with cisplatin resistance are highlighted. Improving the knowledge of the influence of metabolism on cisplatin response is fundamental to identify new possible metabolic targets useful for combinatory treatments, in order to overcome cisplatin resistance.
    Keywords:  Cancer metabolism; Cisplatin; Drug resistance; Glutamine; Glycolysis; Lipid metabolism; Metabolic reprogramming; Metabolic targets; Mitochondria; PPP
  50. Cell Metab. 2020 Jun 02. pii: S1550-4131(20)30249-7. [Epub ahead of print]31(6): 1047-1049
      Mitochondrial fission is sustained through contact with several organelles, including the endoplasmic reticulum, lysosomes, and the actin cytoskeleton. Nagashima et al. (2020) now demonstrate that PI(4)P-containing Golgi-derived vesicles also modulate mitochondrial fission, driven by Arf1 and PI(4)KIIIβ activity, identifying a new organelle contact involved in maintaining mitochondrial homeostasis.