bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒06‒12
34 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Cancer Res. 2022 Jun 08. pii: canres.1301.2021-5-4 10:59:25.103. [Epub ahead of print]
      Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumour suppressors PTEN and SPRY2 (Sprouty 2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumour cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN and SPRY-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumour progression.
  2. Cancers (Basel). 2022 May 25. pii: 2618. [Epub ahead of print]14(11):
      Pancreas ductal adenocarcinoma (PDAC) is one the most aggressive cancers and associated with very high mortality, requiring the development of novel treatments. The mitochondrial voltage-gated potassium channel, Kv1.3 is emerging as an attractive oncologic target but its function in PDAC is unknown. Here, we evaluated the tissue expression of Kv1.3 in resected PDAC from 55 patients using immunohistochemistry (IHC) and show that all tumors expressed Kv1.3 with 60% of tumor specimens having high Kv1.3 expression. In Pan02 cells, the recently developed mitochondria-targeted Kv1.3 inhibitors PCARBTP and PAPTP strongly reduced cell survival in vitro. In an orthotopic pancreas tumor model (Pan02 cells injected into C57BL/6 mice) in immune-competent mice, injection of PAPTP or PCARBTP resulted in tumor reductions of 87% and 70%, respectively. When combined with clinically used Gemcitabine plus Abraxane (albumin-bound paclitaxel), reduction reached 95% and 80% without resultant organ toxicity. Drug-mediated tumor cell death occurred through the p38-MAPK pathway, loss of mitochondrial membrane potential, and oxidative stress. Resistant Pan02 clones to PCARBTP escaped cell death through upregulation of the antioxidant system. In contrast, tumor cells did not develop resistance to PAPTP. Our data show that Kv1.3 is highly expressed in resected human PDAC and the use of novel mitochondrial Kv1.3 inhibitors combined with cytotoxic chemotherapies might be a novel, effective treatment for PDAC.
    Keywords:  Kv1.3; mitochondria; pancreas adenocarcinoma; potassium channel
  3. Cells. 2022 Jun 02. pii: 1827. [Epub ahead of print]11(11):
      O-GlcNAcylation is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. Only two enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase), control the attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells and RAW 264.7 cells, as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a Bioluminescence Resonance Energy Transfer (BRET)-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we show that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA has no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.
    Keywords:  MGEA5; O-GlcNAc transferase; O-GlcNAcase; O-GlcNAcylation; O-linked N-acetylglucosamine transferase; O-linked N-acetylglucosaminidase; hydrogen peroxide; mitochondria; oxidative stress; reactive oxygen species
  4. Genetics. 2022 Jun 06. pii: iyac090. [Epub ahead of print]
      Cytochrome c oxidase (CcO) is a multimeric copper-containing enzyme of the mitochondrial respiratory chain that powers cellular energy production. The two core subunits of CcO, Cox1 and Cox2, harbor the catalytic CuB and CuA sites, respectively. Biogenesis of each copper site occurs separately and requires multiple proteins that constitute the mitochondrial copper delivery pathway. Currently, the identity of all the members of the pathway is not known, though several evolutionarily conserved twin CX9C motif-containing proteins have been implicated in this process. Here, we performed a targeted yeast suppressor screen that placed Coa4, a twin CX9C motif-containing protein, in the copper delivery pathway to the Cox1 subunit. Specifically, we show that overexpression of Cox11, a copper metallochaperone required for the formation of CuB site, can restore Cox1 abundance, CcO assembly, and mitochondrial respiration in coa4Δ cells. This rescue is dependent on the copper-coordinating cysteines of Cox11. The abundance of Coa4 and Cox11 in mitochondria is reciprocally regulated, further linking Coa4 to the CuB site biogenesis. Additionally, we find that coa4Δ cells have reduced levels of copper and exogenous copper supplementation can partially ameliorate its respiratory-deficient phenotype, a finding that connects Coa4 to cellular copper homeostasis. Finally, we demonstrate that human COA4 can replace the function of yeast Coa4 indicating its evolutionarily conserved role. Our work provides genetic evidences for the role of Coa4 in the copper delivery pathway to the CuB site of CcO.
    Keywords:  Coa4; Cox1; Cox11; copper; cytochrome c oxidase; mitochondria
  5. Redox Biol. 2022 Jun 02. pii: S2213-2317(22)00130-6. [Epub ahead of print]54 102358
      The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2•-) -generating drug β-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, β-Lapachone did not generate mitochondrial O2•- but rather SOD2 loss enhanced the efficacy of β-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to β-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.
    Keywords:  KEAP1; NADPH; NFE2L2; NSCLC; ROS; SOD2; β-Lapachone
  6. FEBS Open Bio. 2022 Jun 10.
      Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a "metabolic disorder", is governed by various key regulatory factors like metabolic enzymes, oncogenes and tumor suppressors. Sirtuins (SIRT1-SIRT7) belong to the group of NAD+ deacetylase and ADP-ribosylation enzymes, that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared to the non-malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2 driven activatory phosphorylation of DRP1 at serine-616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA-mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction of stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.
    Keywords:  Aerobic Glycolysis; SIRT6; actin polymerization; mitochondrial fragmentation
  7. Nat Cancer. 2022 Jun 09.
      Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers.
  8. Cell. 2022 May 30. pii: S0092-8674(22)00590-6. [Epub ahead of print]
      The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
    Keywords:  7S RNA; POLRMT; SUV3; cryo-EM; dimer; mitochondria; mtDNA; mtEXO; non-coding RNA; transcription
  9. FEBS Lett. 2022 Jun 03.
      Mitochondria are involved in many cellular activities, including energy metabolism and biosynthesis of nucleotides, fatty acids, and amino acids. Mitochondrial morphology is a key factor in dictating mitochondrial functions. Here, we report that the acyl-CoA binding protein Acb1 in the fission yeast Schizosaccharomyces pombe is required for the maintenance of tubular mitochondrial morphology and proper mitochondrial respiration. The absence of Acb1 causes severe mitochondrial fragmentation in a dynamin-related protein Dnm1-dependent manner and impairs mitochondrial respiration. Moreover, Acb1 regulates the remodeling of lipid droplets in nutrient-rich conditions. Importantly, Acb1 promotes cell survival when cells are cultured in nutrient-rich medium. Hence, our findings establish roles of acyl-CoA binding proteins in regulating mitochondria, lipid droplets, and cell viability.
    Keywords:  Acyl-CoA-Binding Protein; Cell proliferation; Lipid droplets; Mitochondria; Schizosaccharomyces pombe
  10. Leukemia. 2022 Jun 07.
      Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.
  11. Cell Rep. 2022 Jun 07. pii: S2211-1247(22)00689-1. [Epub ahead of print]39(10): 110912
      To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.
    Keywords:  B lymphocyte; CP: Immunology; HIF1; TCA cycle; class switch recombination; germinal center; hypoxia inducible factor 1; mTOR; mammalian target of Rapamycin; mitochondrial DNA; mitochondrial respiration; oxidative phosphorylation; phosphatidic acid; plasma cell
  12. Nat Cell Biol. 2022 Jun 06.
      Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.
  13. Cancers (Basel). 2022 Jun 01. pii: 2750. [Epub ahead of print]14(11):
      Energy metabolism is the basis for cell growth, and cancer cells in particular, are more energy-dependent cells because of rapid cell proliferation. Previously, we found that penfluridol, an antipsychotic drug, has the ability to trigger cell growth inhibition of lung cancer cells via inducing ATP energy deprivation. The toxic effect of penfluridol is related to energy metabolism, but the underlying mechanisms remain unclear. Herein, we discovered that treatment of A549 and HCC827 lung cancer cells with penfluridol caused a decrease in the total amount of ATP, especially in A549 cells. An Agilent Seahorse ATP real-time rate assay revealed that ATP production rates from mitochondrial respiration and glycolysis were, respectively, decreased and increased after penfluridol treatment. Moreover, the amount and membrane integrity of mitochondria decreased, but glycolysis-related proteins increased after penfluridol treatment. Furthermore, we observed that suppression of glycolysis by reducing glucose supplementation or using 2-deoxy-D-glucose (2DG) synergistically enhanced the inhibitory effect of penfluridol on cancer cell growth and the total amount of mitochondria. A mechanistic study showed that the penfluridol-mediated energy reduction was due to inhibition of critical regulators of mitochondrial biogenesis, the sirtuin 1 (SIRT1)/peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) axis. Upregulation of the SIRT1/PGC-1α axis reversed the inhibitory effect of penfluridol on mitochondrial biogenesis and cell viability. Clinical lung cancer samples revealed a positive correlation between PGC-1α (PPARGC1A) and SIRT1 expression. In an orthotopic lung cancer mouse model, the anticancer activities of penfluridol, including growth and metastasis inhibition, were also enhanced by combined treatment with 2DG. Our study results strongly support that a combination of repurposing penfluridol and a glycolysis inhibitor would be a good strategy for enhancing the anticancer activities of penfluridol in lung cancer.
    Keywords:  PGC-1α; SITR1; glycolysis; lung cancer; mitochondria
  14. PLoS One. 2022 ;17(6): e0269620
      Clinical targeting of the altered metabolism of tumor cells has long been considered an attractive hypothetical approach. However, this strategy has yet to perform well clinically. Metabolic redundancy is among the limitations on effectiveness of many approaches, engendering intrinsic single-agent resistance or efficient evolution of such resistance. We describe new studies of the multi-target, tumor-preferential inhibition of the mitochondrial tricarboxylic acid (TCA) cycle by the first-in-class drug CPI-613® (devimistat). By suppressing the TCA hub, indispensable to many metabolic pathways, CPI-613 substantially reduces the effective redundancy of tumor catabolism. This TCA cycle suppression also engenders an apparently homeostatic accelerated, inefficient consumption of nutrient stores in carcinoma cells, eroding some sources of drug resistance. Nonetheless, sufficiently abundant, cell line-specific lipid stores in carcinoma cells are among remaining sources of CPI-613 resistance in vitro and during the in vivo pharmacological drug pulse. Specifically, the fatty acid beta-oxidation step delivers electrons directly to the mitochondrial electron transport system (ETC), by-passing the TCA cycle CPI-613 target and producing drug resistance. Strikingly, tested carcinoma cell lines configure much of this fatty acid flow to initially traverse the peroxisome enroute to additional mitochondrial beta-oxidation. This feature facilitates targeting as clinically practical agents disrupting this flow are available. Two such agents significantly sensitize an otherwise fully CPI-613-resistant carcinoma xenograft in vivo. These and related results are strong empirical support for a potentially general class of strategies for enhanced clinical targeting of carcinoma catabolism.
  15. Cell Rep. 2022 Jun 07. pii: S2211-1247(22)00694-5. [Epub ahead of print]39(10): 110917
      Fumarate can be a surrogate for O2 as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O2 to H2O. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous 13C4-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O2 consumption by uncoupling adjacent RPE mitochondria.
    Keywords:  CP: metabolism; anion transport; cell metabolism; energy metabolism; mitochondrial respiratory chain; retinal metabolism; retinal pigment epithelium; succinate; uncoupling
  16. Front Mol Biosci. 2022 ;9 890402
      Nicotinamide adenine dinucleotide (NAD+) is an essential molecule for living organisms. CD38 is a key NAD+-dependent enzyme which breaks down NAD+ to cyclic ADP-ribose (ADPR) and nicotinamide (NAM, vitamin B3), and NAM can be recycled to synthesize NAD+. CD38 expression is consistently silenced by methylation in prostate cancer and progressively downregulated in advanced castration-resistant prostate cancer, suggesting a connection between NAD+ and prostate carcinogenesis as well as prostate cancer progression. However, the functional interplay between NAD+, CD38, and NAM remains largely uncharacterized in prostate cancer cells. In this study, we generated stable LNCaP95 cell clones expressing varying levels of CD38 upon induction by doxycycline. We demonstrate that CD38 overexpression resulted in growth suppression and apoptosis accompanied by cleavage of poly (ADP-ribose) polymerase 1 (PARP1). CD38 overexpression also dramatically reduced intracellular NAD+ levels and decreased mitochondrial respiration as measured by oxygen consumption rate. We further show that some but not all of these CD38-induced phenotypes could be rescued by exogenous NAM. Treatment of cells with NAM rescued CD38-induced apoptosis and mitochondrial stress but did not restore intracellular NAD+ levels. We also found that NAM demonstrated biphasic effect on mitochondria function, a finding that can be explained by the dual role of NAM as both a precursor of NAD+ and also as a suppressor of a number of NAD+-dependent enzymes. Collectively, these findings provide additional insight supporting the functional relevance of CD38 loss in prostate cancer by linking cell-autonomous regulation of mitochondrial function and prostate cancer.
    Keywords:  CD38; NAD; mitochodria; nicotinamide (NAM); prostate cancer
  17. Exp Cell Res. 2022 Jun 01. pii: S0014-4827(22)00226-9. [Epub ahead of print] 113233
      Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0 cells with puromycin-based selection in the microfluidic device.
    Keywords:  Cell fusion; Cybrid; Microfluidic device; Microtunnel; Mitochondrial transfer
  18. Arch Biochem Biophys. 2022 Jun 02. pii: S0003-9861(22)00117-5. [Epub ahead of print] 109232
      Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.
  19. Commun Biol. 2022 Jun 03. 5(1): 541
      Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.
  20. Natl Sci Rev. 2022 May;9(5): nwab212
      BCAT2-mediated branched-chain amino acid (BCAA) catabolism is critical for pancreatic ductal adenocarcinoma (PDAC) development, especially at an early stage. However, whether a high-BCAA diet promotes PDAC development in vivo, and the underlying mechanism of BCAT2 upregulation, remain undefined. Here, we find that a high-BCAA diet promotes pancreatic intraepithelial neoplasia (PanIN) progression in LSL-KrasG12D/+ ; Pdx1-Cre (KC) mice. Moreover, we screened with an available deubiquitylase library which contains 31 members of USP family and identified that USP1 deubiquitylates BCAT2 at the K229 site. Furthermore, BCAA increases USP1 protein at the translational level via the GCN2-eIF2α pathway both in vitro and in vivo. More importantly, USP1 inhibition recedes cell proliferation and clone formation in PDAC cells and attenuates pancreas tumor growth in an orthotopic transplanted mice model. Consistently, a positive correlation between USP1 and BCAT2 is found in KC; LSL-KrasG12D/+ ; p53flox/+ ; Pdx1-Cre mice and clinical samples. Thus, a therapeutic targeting USP1-BCAT2-BCAA metabolic axis could be considered as a rational strategy for treatment of PDAC and precisive dietary intervention of BCAA has potentially translational significance.
    Keywords:  BCAT2; PDAC; PanIN; USP1; deubiquitylation
  21. Cells. 2022 May 24. pii: 1731. [Epub ahead of print]11(11):
      Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a certain level of ETC dysfunction is reached. Cells can invoke mechanisms to circumvent reaching their critical ETC threshold, but it is an ongoing challenge to identify such processes. In the nematode Caenorhabditis elegans, severe reduction of mitochondrial ETC activity shortens life, but mild reduction actually extends it, providing an opportunity to identify threshold circumvention mechanisms. Here, we show that removal of ATL-1, but not ATM-1, worm orthologs of ATR and ATM, respectively, key nuclear DNA damage checkpoint proteins in human cells, unexpectedly lessens the severity of ETC dysfunction. Multiple genetic and biochemical tests show no evidence for increased mutation or DNA breakage in animals exposed to ETC disruption. Reduced ETC function instead alters nucleotide ratios within both the ribo- and deoxyribo-nucleotide pools, and causes stalling of RNA polymerase, which is also known to activate ATR. Unexpectedly, atl-1 mutants confronted with mitochondrial ETC disruption maintain normal levels of oxygen consumption, and have an increased abundance of translating ribosomes. This suggests checkpoint signaling by ATL-1 normally dampens cytoplasmic translation. Taken together, our data suggest a model whereby ETC insufficiency in C. elegans results in nucleotide imbalances leading to the stalling of RNA polymerase, activation of ATL-1, dampening of global translation, and magnification of ETC dysfunction. The loss of ATL-1 effectively reverses the severity of ETC disruption so that animals become phenotypically closer to wild type.
    Keywords:  DDR; DNA damage response; MAK-1; MAK-2; MAPKAPs; Mit mutants; ageing; aging; checkpoint response; polysome profiling; retrograde response
  22. Bioorg Chem. 2022 May 20. pii: S0045-2068(22)00261-9. [Epub ahead of print]126 105856
      Tumor-necrosis-factor-receptor associated protein 1 (TRAP1), a mitochondrial paralog of heat shock protein 90 family proteins, is overexpressed in many cancer cells and supports tumorigenesis by rewiring vital metabolic and cell death pathways. The triphenylphosphonium moiety is used to deliver therapeutic cargo to increase drug uptake into mitochondria. Various aryl- or alkyl-substituted phosphonium analogs were conjugated with TRAP1-selective inhibitors 4a-c to optimize anticancer activity. Among these various phosphonium-conjugated compounds, (6-(2-amino-9-(4-bromo-2-fluorobenzyl)-6-chloro-8-oxo-8,9-dihydro-7H-purin-7-yl)hexyl)triphenylphosphornium (6a) was identified as a potential anticancer agent. Compound 6a had IC50 values of 0.30-3.24 μM in seven different cancer cell lines and potently suppressed tumor growth without any noticeable in vivo toxicity in a nude mouse model xenografted with PC3 prostate cancer cells.
    Keywords:  Hsp90; Inhibitor; Mitochondria; TRAP1; Triphenylphosphonium
  23. iScience. 2022 Jun 17. 25(6): 104468
      The sharp increase in obesity prevalence worldwide is mainly attributable to changes in physical activity and eating behavior but the metabolic and clinical impacts of these obesogenic conditions vary between sexes and genetic backgrounds. This warrants personalized treatments of obesity and its complications, which require a thorough understanding of the diversity of metabolic responses to high-fat diet intake. By analyzing nine genetically diverse mouse strains, we show that much like humans, mice exhibit a huge variety of physiological and biochemical responses to high-fat diet. The strains exhibit various degrees of alterations in their phenotypic makeup. At the transcriptome level, we observe dysregulations of immunity, translation machinery, and mitochondrial genes. At the biochemical level, the enzymatic activity of mitochondrial complexes is affected. The diversity across mouse strains, diets, and sexes parallels that found in humans and supports the use of diverse mouse populations in future mechanistic or preclinical studies on metabolic dysfunctions.
    Keywords:  Biological sciences; Endocrinology; Obesity medicine
  24. J Exp Biol. 2022 Jun 08. pii: jeb.243680. [Epub ahead of print]
      This study asked whether interindividual variation in maximum and standard aerobic metabolic rates of the Gulf killifish, Fundulus grandis, correlate with gill morphology and cardiac mitochondrial bioenergetics, traits reflecting critical steps in the O2 transport cascade from the environment to the tissues. Maximum metabolic rate (MMR) was positively related to body mass, total gill filament length, and myocardial oxygen consumption during maximum oxidative phosphorylation (multiple R2=0.836). Standard metabolic rate (SMR) was positively related to body mass, total gill filament length, and myocardial oxygen consumption during maximum electron transport system activity (multiple R2=0.717). After controlling for body mass, individuals with longer gill filaments, summed over all gill arches, or greater cardiac respiratory capacity had higher whole-animal metabolic rates. The overall model fit and the explanatory power of individual predictor variables were better for MMR than for SMR, suggesting that gill morphology and myocardial bioenergetics are more important in determining active rather than resting metabolism. After accounting for body mass, heart ventricle mass was not related to variation in MMR or SMR, indicating that the quality of the heart (i.e., the capacity for mitochondrial metabolism) was more influential than heart size. Finally, the myocardial oxygen consumption required to offset the dissipation of the transmembrane proton gradient in the absence of ATP synthesis was not correlated with either MMR or SMR. The results support the idea that interindividual variation in aerobic metabolism, particularly maximum metabolic rate, is associated with variation in specific steps in the O2 transport cascade.
    Keywords:  Fundulus; Gill morphology; Heart; Metabolic rate; Mitochondria; Oxygen transport cascade
  25. Biochim Biophys Acta Mol Basis Dis. 2022 Jun 06. pii: S0925-4439(22)00125-9. [Epub ahead of print] 166455
      Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We showed that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment which may lead to cell death of breast cancer cells/BCSCs.
    Keywords:  Autophagic flux; Autophagy; Breast Cancer stem cells; Breast cancer; Energy metabolism
  26. Nat Cancer. 2022 Jun 06.
      Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.
  27. Aging Cell. 2022 Jun 03. e13647
      Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD+ ) declining over time. The decrease in NAD+ production has been linked to the age-related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate-limiting enzyme in the NAD+ biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV-eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD+ and SIRT1 activity in recipient cells compared to pre-exercise EVs, indicating a pathway for inter-tissue signalling promoted through exercise. Our results suggest a mechanism to limit age-related NAD+ decline, through the systemic delivery of eNAMPT via EVs released during exercise.
    Keywords:  NAD+; NAMPT; SIRT1; aging; exercise; exosome; extracellular vesicles; healthspan
  28. Autophagy. 2022 Jun 05. 1-2
      The protein TRIM5 is under intensive investigation related to its roles in antiviral defense, yet its underlying mechanisms of action remain elusive. In our study, we performed an unbiased identification of TRIM5-interacting partners and found proteins participating in a wide variety of cellular functions. We utilized this proteomics data set to uncover a role for TRIM5 in mitophagy, a mitochondrial quality control system that is impaired in multiple human diseases. Mitochondrial damage triggers the recruitment of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalizes with markers of autophagosome biogenesis. Cells lacking TRIM5 are unable to carry out PRKN-dependent and PRKN-independent mitophagy pathways. TRIM5 knockout cells show reduced mitochondrial function and uncontrolled immune activation in response to mitochondrial damage; phenotypes consistent with a requirement for TRIM5 in mitophagy. Mechanistically, we found that TRIM5 is required for the recruitment of the autophagy initiation machinery to damaged mitochondria, where TRIM5 acts as a scaffold promoting interactions between protein markers of mitochondrial damage and the autophagy initiation machinery.
    Keywords:  APEX2; HIV-1; TRIM5α; autophagy; inflammation; mitochondria; mitophagy; restriction factor; tripartite-motif
  29. Elife. 2022 Jun 08. pii: e75426. [Epub ahead of print]11
      Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP properties such as size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.
    Keywords:  S. cerevisiae; biochemistry; chemical biology
  30. Cancer Med. 2022 Jun 06.
      BACKGROUND: The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, with deregulation leading to cancer and other diseases. However, how this pathway is dysregulated in cancer has not been well clarified.METHODS: Using a tandem affinity purification/mass-spec technique and biochemical analyses, we identified tumor protein D52 (TPD52) as an AMPKα-interacting molecule. To explore the biological effects of TPD52 in cancers, we conducted biochemical and metabolic assays in vitro and in vivo with cancer cells and TPD52 transgenic mice. Finally, we assessed the clinical significance of TPD52 expression in breast cancer patients using bioinformatics techniques.
    RESULTS: TPD52, initially identified to be overexpressed in many human cancers, was found to form a stable complex with AMPK in cancer cells. TPD52 directly interacts with AMPKα and inhibits AMPKα kinase activity in vitro and in vivo. In TPD52 transgenic mice, overexpression of TPD52 leads to AMPK inhibition and multiple metabolic defects. Clinically, high TPD52 expression predicts poor survival of breast cancer patients.
    CONCLUSION: The findings revealed that TPD52 is a novel regulator of energy stress-induced AMPK activation and cell metabolism. These results shed new light on AMPK regulation and understanding of the etiology of cancers with TPD52 overexpression.
    Keywords:  AMP-activated protein kinase (AMPK); cell metabolism; tumor protein D52 (TPD52)
  31. PLoS One. 2022 ;17(6): e0269432
      Clear cell renal cell carcinoma (ccRCC) alters metabolic signals frequently, leading to mitochondrial dysfunction, such as increase of glycolysis and accumulation of lipid. Sirtuin3 (SIRT3) is a key factor for the regulation of both mitochondrial integrity and function. SIRT3 is downregulated and contributes in both cancer development and progression in ccRCC. The aim of this study is to investigate SIRT3-regulated mitochondrial biogenesis in ccRCC. SIRT3 overexpression alone reduced glucose uptake rate and enhanced membrane potential in mitochondria. ccRCC with overexpressed SIRT3 further improved the lethal effects when combined with anticancer drugs (Resveratrol, Everolimus and Temsirolimus). Cell viability was markedly decreased in a dose-dependent manner when treated with resveratrol or mTOR inhibitors in SIRT3 overexpressing ccRCC. In conclusion, SIRT3 improved mitochondrial functions in ccRCC through metabolic reprogramming. Mitochondrial reprogramming by SIRT3 regulation improves the sensitivity to anticancer drugs. The combination of SIRT3 and resveratrol functioned synergistically lethal effect in ccRCC.
  32. Mol Oncol. 2022 Jun 05.
      Aging is the most robust risk factor for cancer development, with more than 60% of cancers occurring in those aged 60 and above. However, how aging and tumorigenesis are intertwined is poorly understood and a matter of significant debate. Metabolic changes are hallmarks of both aging and tumorigenesis. The deleterious consequences of aging include dysfunctional cellular processes, the build up of metabolic by-products and waste molecules in circulation and within tissues, and stiffer connective tissues that impede blood flow and oxygenation. Collectively, these age-driven changes lead to metabolic reprogramming in different cell types of a given tissue that significantly affects their cellular functions. Here, we put forward the idea that metabolic changes that happen during aging help create a favorable environment for tumorigenesis. We review parallels in metabolic changes that happen during aging and how these changes function both as adaptive mechanisms that enable the development of malignant phenotypes in a cell-autonomous manner and as mechanisms that suppress immune surveillance, collectively creating the perfect environment for cancers to thrive. Hence, anti-aging therapeutic strategies that target the metabolic reprogramming that occurs as we age might provide new opportunities to prevent cancer initiation and/or improve responses to standard-of-care anti-cancer therapies.
    Keywords:  Aging; cellular energetics; immune response; metabolic reprogramming; tumorigenesis
  33. J Ovarian Res. 2022 Jun 07. 15(1): 70
      BACKGROUND: Mitochondrial dynamics (e.g. fission/fusion) play an important role in controlling chemoresistance in representative gynecologic malignancies, ovarian and cervical cancer. Processing the long form of Optic atrophy (L-Opa)1 is a distinctive character of mitochondrial fragmentation, associated with chemosensitivity. Here, we examined the role of prohibitin (Phb)1 in increasing L-Opa1 processing via the regulating mitochondrial protease, Oma1 and its direct interaction with p-p53 (ser15) and pro-apoptotic Bcl-2 antagonist/killer (Bak) 1 in the signaling axis and if this phenomenon is associated with prognosis of patients.METHODS: We compared Cisplatin (CDDP)-induced response of mitochondrial dynamics, molecular interaction among p-p53 (ser15)-Phb1-Bak, and chemoresponsiveness in paired chemosensitive and chemoresistant gynecologic cancer cells (ovarian and cervical cancer cell lines) using western blot, immunoprecipitation, sea horse, and immunofluorescence. Translational strategy with proximity ligation assessment in phb1-p-p53 (ser15) in human ovarian tumor sections further confirmed in vitro finding, associated with clinical outcome.
    RESULTS: We report that: (1) Knock-down of Phb1 prevents Cisplatin (cis-diamine-dichloroplatinum; CDDP) -induced changes in mitochondrial fragmentation and Oma1 mediated cleavage, and Opa1 processing; (2) In response to CDDP, Phb1 facilitates the p-p53 (ser15)-Phb1-Bak interaction in mitochondria in chemosensitive gynecologic cancer cells but not in chemoresistant cells; (3) Akt overexpression results in suppressed p-p53(Ser15)-Phb1 interaction and dysregulated mitochondrial dynamics, and (4) Consistent with in vitro findings, proximity ligation assessment (PLA) in human ovarian tumor sections demonstrated that p-p53(ser15)-Phb1-Bak interaction in mitochondria is associated with better chemoresponsiveness and clinical outcome of patients. Determining the molecular mechanisms by which Phb1 facilitates mitochondrial fragmentation and interacts with p53 may advance the current understanding of chemoresistance and pathogenesis of gynecologic cancer.
    CONCLUSION: Determining the key molecular mechanisms by which Phb1 facilitates the formation of p-p53 (ser15)-Bak-Phb1 and its involvement in the regulation of mitochondrial dynamics and apoptosis may ultimately contribute to the current understanding of molecular and cellular basis of chemoresistance in this gynecologic cancer.
    Keywords:  Bak; CDDP; Chemoresistance; Mitochondrial fragmentation; Phb1; p53