bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒08‒22
forty-five papers selected by
Kelsey Fisher-Wellman, East Carolina University

  1. Front Oncol. 2021 ;11 678343
      MitoTracker Deep Red (MTDR) is a relatively non-toxic, carbocyanine-based, far-red, fluorescent probe that is routinely used to chemically mark and visualize mitochondria in living cells. Previously, we used MTDR at low nano-molar concentrations to stain and metabolically fractionate breast cancer cells into Mito-high and Mito-low cell sub-populations, by flow-cytometry. Functionally, the Mito-high cell population was specifically enriched in cancer stem cell (CSC) activity, i) showing increased levels of ESA cell surface expression and ALDH activity, ii) elevated 3D anchorage-independent growth, iii) larger overall cell size (>12-μm) and iv) Paclitaxel-resistance. The Mito-high cell population also showed enhanced tumor-initiating activity, in an in vivo preclinical animal model. Here, we explored the hypothesis that higher nano-molar concentrations of MTDR could also be used to therapeutically target and eradicate CSCs. For this purpose, we employed an ER(+) cell line (MCF7) and two triple negative cell lines (MDA-MB-231 and MDA-MB-468), as model systems. Remarkably, MTDR inhibited 3D mammosphere formation in MCF7 and MDA-MB-468 cells, with an IC-50 between 50 to 100 nM; similar results were obtained in MDA-MB-231 cells. In addition, we now show that MTDR exhibited near complete inhibition of mitochondrial oxygen consumption rates (OCR) and ATP production, in all three breast cancer cell lines tested, at a level of 500 nM. However, basal glycolytic rates in MCF7 and MDA-MB-468 cells remained unaffected at levels of MTDR of up to 1 μM. We conclude that MTDR can be used to specifically target and eradicate CSCs, by selectively interfering with mitochondrial metabolism, by employing nano-molar concentrations of this chemical entity. In further support of this notion, MTDR significantly inhibited tumor growth and prevented metastasis in vivo, in a xenograft model employing MDA-MB-231 cells, with little or no toxicity observed. In contrast, Abemaciclib, an FDA-approved CDK4/6 inhibitor, failed to inhibit metastasis. Therefore, in the future, MTDR could be modified and optimized via medicinal chemistry, to further increase its potency and efficacy, for its ultimate clinical use in the metabolic targeting of CSCs for their eradication.
    Keywords:  MitoTracker Deep Red (MTDR); anti-metastatic activity; anti-tumor activity; cancer stem-like cells (CSCs); cancer therapy; mitochondria; near-infrared dyes (NIR)
  2. Antiviral Res. 2021 Aug 11. pii: S0166-3542(21)00149-2. [Epub ahead of print]194 105159
      Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV requires functional host cell mitochondria for efficient replication, it is unknown whether mitochondrial targeted pharmacological agents can be repurposed as antivirals. Here we report that treatment with drugs targeting the electron transport chain (ETC) complexes inhibit HCMV replication. Addition of rotenone, oligomycin, antimycin and metformin resulted in decreased HCMV titers in vitro, independent of HCMV strain. This further illustrates the dependence of HCMV replication on functional mitochondria. Metformin, an FDA approved drug, delays HCMV replication kinetics resulting in a reduction of viral titers. Repurposing metformin as an antiviral is advantageous as its safety profile and epidemiological data are well accepted. Our findings provide new insight into the potential for targeting HCMV infection through host cell metabolism and how these pharmacological interventions function.
    Keywords:  CMV; Cytomegalovirus; Electron transport chain; Metformin; Mitochondria
  3. Curr Res Physiol. 2021 ;4 163-176
      Folding of the mitochondrial inner membrane (IM) into cristae greatly increases the ATP-generating surface area, S IM, per unit volume but also creates diffusional bottlenecks that could limit reaction rates inside mitochondria. This study explores possible effects of inner membrane folding on mitochondrial ATP output, using a mathematical model for energy metabolism developed by the Jafri group and two- and three-dimensional spatial models for mitochondria, implemented on the Virtual Cell platform. Simulations demonstrate that cristae are micro-compartments functionally distinct from the cytosol. At physiological steady states, standing gradients of ADP form inside cristae that depend on the size and shape of the compartments, and reduce local flux (rate per unit area) of the adenine nucleotide translocase. This causes matrix ADP levels to drop, which in turn reduces the flux of ATP synthase. The adverse effects of membrane folding on reaction fluxes increase with crista length and are greater for lamellar than tubular crista. However, total ATP output per mitochondrion is the product of flux of ATP synthase and S IM which can be two-fold greater for mitochondria with lamellar than tubular cristae, resulting in greater ATP output for the former. The simulations also demonstrate the crucial role played by intracristal kinases (adenylate kinase, creatine kinase) in maintaining the energy advantage of IM folding.
    Keywords:  ATP synthesis; Computational modeling; Cristae; Energy metabolism; Kinases; Mitochondria
  4. Commun Biol. 2021 Aug 19. 4(1): 989
    LSFC Consortium
      Mouse models of genetic mitochondrial disorders are generally used to understand specific molecular defects and their biochemical consequences, but rarely to map compensatory changes allowing survival. Here we took advantage of the extraordinary mitochondrial resilience of hepatic Lrpprc knockout mice to explore this question using native proteomics profiling and lipidomics. In these mice, low levels of the mtRNA binding protein LRPPRC induce a global mitochondrial translation defect and a severe reduction (>80%) in the assembly and activity of the electron transport chain (ETC) complex IV (CIV). Yet, animals show no signs of overt liver failure and capacity of the ETC is preserved. Beyond stimulation of mitochondrial biogenesis, results show that the abundance of mitoribosomes per unit of mitochondria is increased and proteostatic mechanisms are induced in presence of low LRPPRC levels to preserve a balance in the availability of mitochondrial- vs nuclear-encoded ETC subunits. At the level of individual organelles, a stabilization of residual CIV in supercomplexes (SCs) is observed, pointing to a role of these supramolecular arrangements in preserving ETC function. While the SC assembly factor COX7A2L could not contribute to the stabilization of CIV, important changes in membrane glycerophospholipid (GPL), most notably an increase in SC-stabilizing cardiolipins species (CLs), were observed along with an increased abundance of other supramolecular assemblies known to be stabilized by, and/or participate in CL metabolism. Together these data reveal a complex in vivo network of molecular adjustments involved in preserving mitochondrial integrity in energy consuming organs facing OXPHOS defects, which could be therapeutically exploited.
  5. Proc Natl Acad Sci U S A. 2021 Aug 24. pii: e2101674118. [Epub ahead of print]118(34):
      The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.
    Keywords:  metabolism; mitochondria; reactive oxygen species
  6. Mol Cell Biol. 2021 Aug 16. MCB0023321
      Mitochondrial oxidative phosphorylation (OXPHOS) enzymes are made up of dual genetic origin. Mechanisms regulating the expression of nuclear-encoded OXPHOS subunits in response to metabolic cues (glucose vs. glycerol), is significantly understood while regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3 a DEAD/H box helicase, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunit in cells consistent with its role in regulating translation elongation based on Arg8m reporter system. IRC3 deleted cells retained mitochondrial DNA despite growth defect on glycerol plates. Glucose grown Δirc3ρ+ and irc3 temperature-sensitive cells at 370C have reduced translation rates from majority of mRNAs. In contrast, when galactose was the carbon source, reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ+ but no defect was observed in irc3 temperature-sensitive cells, at 370C. In support, of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, suppressors of Δirc3 isolated for restoration of growth on glycerol media restore mitochondrial protein synthesis differentially in presence of glucose vs. glycerol.
  7. Clin Cancer Res. 2021 Aug 18. pii: clincanres.4789.2020. [Epub ahead of print]
      PURPOSE: Human papillomavirus (HPV) infection drives the development of some head and neck cancer squamous cell carcinomas (HNSCC). This disease is rapidly increasing in incidence worldwide. Although these tumors are sensitive to treatment, ~10% of patients fail therapy. However, the mechanisms that underlie treatment failure remain unclear.EXPERIMENTAL DESIGN: We performed RNA seq on tissues from matched primary (pOPSCC) and metachronous recurrent cancers (rOPSCC) to identify transcriptional differences to gain mechanistic insight into the evolutionary adaptations of metachronous recurrent tumors. We used HPV-related HNSCC cells lines to investigate the effect of (1) NRF2 overexpression on growth in vitro and in vivo (2) OXPHOS inhibition using IACS-010759 on NRF2 dependent cells (3) combination of cisplatin and OXPHOS inhibition.
    RESULTS: The oxidative phosphorylation (OXPHOS) pathway is enriched in recurrent HPV-associated HNSCC and may contribute to treatment failure. NRF2-enriched HNSCC samples from the Cancer Genome Atlas with enrichment in OXPHOS, fatty acid metabolism, Myc, Mtor, ROS, and glycolytic signaling networks exhibited worse survival. HPV-positive HNSCC cells demonstrated sensitivity to the OXPHOS inhibitor, in a NRF2-dependent manner. Further, using murine xenograft models, we identified NRF2 as a driver of tumor growth. Mechanistically, NRF2 drives ROS and mitochondrial respiration, and NRF2 is a critical regulator of redox homeostasis that can be crippled by disruption of OXPHOS. NRF2 also mediated cisplatin sensitivity in endogenously overexpressing primary HPV-related HNSCC cells.
    CONCLUSIONS: These results unveil a paradigm shifting translational target harnessing NRF2-mediated metabolic reprogramming in HPV-related HNSCC.
  8. Bio Protoc. 2021 Jul 20. 11(14): e4087
      The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.
    Keywords:   Ca2+; Anti-neoplastic strategy; Cancer; Cell-penetrating peptide; Hexokinase 2; Mitochondria; Mitochondria-associated membranes
  9. Transl Res. 2021 Aug 16. pii: S1931-5244(21)00193-6. [Epub ahead of print]
    TAIPAI Study Group
      Elevated serum aldosterone promotes arterial hypertension, cardiac hypertrophy, and diastolic dysfunction. However, the effect of elevated aldosterone levels on cardiac mitochondria remains unclear. We used primary cultures of mouse cardiomyocytes to determine whether aldosterone has direct effects on cardiomyocyte mitochondria, and aldosterone-infused mice as a preclinical model to evaluate the impact of aldosterone in vivo. We show that aldosterone suppressed mtDNA copy number and SOD2 expression via the mineralocorticoid receptor (MR)-dependent regulation of NADPH oxidase 2 (NOX2) and generation of reactive oxygen species (ROS) in primary mouse cardiomyocytes. Aldosterone suppressed cardiac mitochondria ATP production, which was rescued by N-acetylcysteine. Aldosterone infusion for 4 weeks in mice suppressed the number of cardiac mitochondria, mtDNA copy number, and SOD2 protein expression. MR blockade by eplerenone or the administration of N-acetylcysteine prevented aldosterone-induced cardiac mitochondrial damage in vivo. Similarly, patients with primary aldosteronism had a lower plasma leukocyte mtDNA copy number. Plasma leukocyte mtDNA copy number was positively correlated with 24-hour urinary aldosterone level and left ventricular mass index. In conclusion, aldosterone suppresses cardiac mitochondria in vivo and directly via MR activation of ROS pathways.
    Keywords:  NOX2; ROS; aldosterone; mitochondria
  10. Diabetologia. 2021 Aug 14.
      AIMS/HYPOTHESIS: This study interrogated mitochondrial respiratory function and content in skeletal muscle biopsies of healthy adults between 30 and 72 years old with and without uncomplicated type 1 diabetes.METHODS: Participants (12 women/nine men) with type 1 diabetes (48 ± 11 years of age), without overt complications, were matched for age, sex, BMI and level of physical activity to participants without diabetes (control participants) (49 ± 12 years of age). Participants underwent a Bergström biopsy of the vastus lateralis to assess mitochondrial respiratory function using high-resolution respirometry and citrate synthase activity. Electron microscopy was used to quantify mitochondrial content and cristae (pixel) density.
    RESULTS: Mean mitochondrial area density was 27% lower (p = 0.006) in participants with type 1 diabetes compared with control participants. This was largely due to smaller mitochondrial fragments in women with type 1 diabetes (-18%, p = 0.057), as opposed to a decrease in the total number of mitochondrial fragments in men with diabetes (-28%, p = 0.130). Mitochondrial respiratory measures, whether estimated per milligram of tissue (i.e. mass-specific) or normalised to area density (i.e. intrinsic mitochondrial function), differed between cohorts, and demonstrated sexual dimorphism. Mass-specific mitochondrial oxidative phosphorylation (OXPHOS) capacity with the substrates for complex I and complex II (CI + II) was significantly lower (-24%, p = 0.033) in women with type 1 diabetes compared with control participants, whereas mass-specific OXPHOS capacities with substrates for complex I only (pyruvate [CI pyr] or glutamate [CI glu]) or complex II only (succinate [CII succ]) were not different (p > 0.404). No statistical differences (p > 0.397) were found in mass-specific OXPHOS capacity in men with type 1 diabetes compared with control participants despite a 42% non-significant increase in CI glu OXPHOS capacity (p = 0.218). In contrast, intrinsic CI + II OXPHOS capacity was not different in women with type 1 diabetes (+5%, p = 0.378), whereas in men with type 1 diabetes it was 25% higher (p = 0.163) compared with control participants. Men with type 1 diabetes also demonstrated higher intrinsic OXPHOS capacity for CI pyr (+50%, p = 0.159), CI glu (+88%, p = 0.033) and CII succ (+28%, p = 0.123), as well as higher intrinsic respiratory rates with low (more physiological) concentrations of either ADP, pyruvate, glutamate or succinate (p < 0.012). Women with type 1 diabetes had higher (p < 0.003) intrinsic respiratory rates with low concentrations of succinate only. Calculated aerobic fitness (Physical Working Capacity Test [PWC130]) showed a strong relationship with mitochondrial respiratory function and content in the type 1 diabetes cohort.
    CONCLUSIONS/INTERPRETATION: In middle- to older-aged adults with uncomplicated type 1 diabetes, we conclude that skeletal muscle mitochondria differentially adapt to type 1 diabetes and demonstrate sexual dimorphism. Importantly, these cellular alterations were significantly associated with our metric of aerobic fitness (PWC130) and preceded notable impairments in skeletal mass and strength.
    Keywords:  Aerobic fitness; Mitochondria; Older adults; Oxidative phosphorylation; Skeletal muscle; Type 1 diabetes
  11. J Natl Cancer Inst. 2021 Aug 20. pii: djab158. [Epub ahead of print]
      BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II; CII), a known tumor suppressor in PPGL.METHODS: A cohort of 352 subjects with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2.
    RESULTS: We describe eight germline variants in the GTP-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of CII and faulty assembly of the complex, resulting in aberrant respiration and elevated succinate accumulation.
    CONCLUSIONS: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.
  12. Mol Cancer Ther. 2021 Aug 19. pii: molcanther.MCT-20-0652-A.2020. [Epub ahead of print]
      Pancreatic Ductal Adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA approved anti-helminthic Pyrvinium Pamoate (PP) has been previously shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50s (9-93nM) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; p<0.0001) and oral administration (PO; p=0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (p<0.001) leading to an increase in glycolysis (p<0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect which was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB approved window of opportunity trial (IND:144822).
  13. J Cell Mol Med. 2021 Aug 17.
      Astragaloside IV (AST) is a major bioactive compound of Radix Astragali with medical and health benefits. Previous studies have found that AST can reduce the body weights of high-fat diet fed mice. However, the effect of AST on fat metabolism of ageing mice is unclear. In this study, naturally ageing mice were administered intragastrically with AST at 30 mg/kg/day (ageing + AST-L group) and 90 mg/kg/day (ageing + AST-H group) for 16-20 months. Adult (4 months old) and ageing mice were given 1% sodium carboxyl methylcellulose as vehicle. Energy metabolism-related biological parameters of living mice were examined. Moreover, mRNA and protein levels of key enzymes/proteins involved in triglyceride (TG) lipolysis, fatty acid β-oxidation (FAO), ketone body (KB) production and mitochondrial respiratory chain were also examined after sacrifice. Results demonstrated that treatment with AST significantly reduced body weight, white fat and liver/body weight ratio of ageing mice, significantly reduced serum/hepatic TG levels, respiratory quotient, promoted fatty acid mobilization in white adipose tissue, mitochondrial FAO and KB production and mitochondrial biosynthesis/functions in the liver of ageing mice. AST also up-regulated the expression of phosphorylated AMP-activated protein kinase, acetyl-CoA carboxylase, acetyl-coenzyme A synthetase, carnitine palmitoyltransferase 1a/1b, enoyl coenzyme A hydratase-short chain, acyl-CoA dehydrogenase medium chain and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase-2 involved in fat metabolism. These results indicated that mitochondrial activity could be the target of AST to treat abnormal fat metabolism during ageing.
    Keywords:  Astragaloside IV; ageing; energy metabolism; fat catabolism; mitochondria; respiratory quotient; β-oxidation
  14. Trends Cancer. 2021 Aug 17. pii: S2405-8033(21)00162-X. [Epub ahead of print]
      Focus on metabolic reprogramming has re-emerged in recent years due to the far-reaching consequences of metabolism on nearly all cellular behaviors. In a recent study in Cell Metabolism, Tharp et al. show that adhesion-dependent mechanical signaling induces mitochondrial and metabolic reprogramming to help cells adapt to future oxidative stress.
    Keywords:  ROS; matrix stiffness; mechanosignaling; mitohormesis
  15. Cell Rep. 2021 Aug 17. pii: S2211-1247(21)00993-1. [Epub ahead of print]36(7): 109559
      Acute myeloid leukemia (AML) is a rapidly progressing cancer, for which chemotherapy remains standard treatment and additional therapeutic targets are requisite. Here, we show that AML cells secrete the stem cell growth factor R-spondin 2 (RSPO2) to promote their self-renewal and prevent cell differentiation. Although RSPO2 is a well-known WNT agonist, we reveal that it maintains AML self-renewal WNT independently, by inhibiting BMP receptor signaling. Autocrine RSPO2 signaling is also required to prevent differentiation and to promote self-renewal in normal hematopoietic stem cells as well as primary AML cells. Comprehensive datamining reveals that RSPO2 expression is elevated in patients with AML of poor prognosis. Consistently, inhibiting RSPO2 prolongs survival in AML mouse xenograft models. Our study indicates that in AML, RSPO2 acts as an autocrine BMP antagonist to promote cancer cell renewal and may serve as a marker for poor prognosis.
    Keywords:  BMP; HSPC; R-spondin; WNT; acute myeloid leukemia; drug resistance; leukemia stem cell; macrophages; monocytes; self-renewal
  16. Cell Death Differ. 2021 Aug 20.
      The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid β-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid β-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.
  17. Nat Methods. 2021 Aug 19.
      Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding mitochondrial form and function.
  18. iScience. 2021 Aug 20. 24(8): 102895
      The mitochondrial uniporter is a Ca2+-selective ion-conducting channel in the inner mitochondrial membrane that is involved in various cellular processes. The components of this uniporter, including the pore-forming membrane subunit MCU and the modulatory subunits MCUb, EMRE, MICU1, and MICU2, have been identified in recent years. Previously, extensive studies revealed various aspects of uniporter activities and proposed multiple regulatory models of mitochondrial Ca2+ uptake. Recently, the individual auxiliary components of the uniporter and its holocomplex have been structurally characterized, providing the first insight into the component structures and their spatial relationship within the context of the uniporter. Here, we review recent uniporter structural studies in an attempt to establish an architectural framework, elucidating the mechanism that governs mitochondrial Ca2+ uptake and regulation, and to address some apparent controversies. This information could facilitate further characterization of mitochondrial Ca2+ permeation and a better understanding of uniporter-related disease conditions.
    Keywords:  Ion; Membranes; Molecular biology
  19. Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00038-1. [Epub ahead of print]363 49-121
      Cancer is a leading cause of death worldwide. All major tumor suppressors and oncogenes are now recognized to have fundamental connections with metabolic pathways. A hallmark feature of cancer cells is a reprogramming of their metabolism even when nutrients are available. Increasing evidence indicates that most cancer cells rely on mitochondrial metabolism to sustain their energetic and biosynthetic demands. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca2+) storage organelle in mammalian cells, through special domains known as mitochondria-ER contact sites (MERCS). In this domain, the release of Ca2+ from the ER is mainly regulated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), a family of Ca2+ release channels activated by the ligand IP3. IP3R mediated Ca2+ release is transferred to mitochondria through the mitochondrial Ca2+ uniporter (MCU). Once in the mitochondrial matrix, Ca2+ activates several proteins that stimulate mitochondrial performance. The role of IP3R and MCU in cancer, as well as the other proteins that enable the Ca2+ communication between these two organelles is just beginning to be understood. Here, we describe the function of the main players of the ER mitochondrial Ca2+ communication and discuss how this particular signal may contribute to the rise and development of cancer traits.
    Keywords:  Cancer hallmarks; IP3R; MCUC; Metabolism; Mitochondria-associated membranes (MAMs)
  20. Proc Biol Sci. 2021 Aug 25. 288(1957): 20211585
      Doubly uniparental inheritance (DUI) represents a notable exception to the general rule of strict maternal inheritance (SMI) of mitochondria in metazoans. This system entails the coexistence of two mitochondrial lineages (F- and M-type) transmitted separately through oocytes and sperm, thence providing an unprecedented opportunity for the mitochondrial genome to evolve adaptively for male functions. In this study, we explored the impact of a sex-specific mitochondrial evolution upon gamete bioenergetics of DUI and SMI bivalve species, comparing the activity of key enzymes of glycolysis, fermentation, fatty acid metabolism, tricarboxylic acid cycle, oxidative phosphorylation and antioxidant metabolism. Our findings suggest reorganized bioenergetic pathways in DUI gametes compared to SMI gametes. This generally results in a decreased enzymatic capacity in DUI sperm with respect to DUI oocytes, a limitation especially prominent at the terminus of the electron transport system. This bioenergetic remodelling fits a reproductive strategy that does not require high energy input and could potentially link with the preservation of the paternally transmitted mitochondrial genome in DUI species. Whether this phenotype may derive from positive or relaxed selection acting on DUI sperm is still uncertain.
    Keywords:  DUI; bioenergetics; bivalves; gametes; mitochondria
  21. Radiat Res. 2021 Aug 16.
      Nicotinamide phosphoribosyltransferase (NAMPT) is the key rate-limiting enzyme in the regulation of nicotinamide adenine dinucleotide (NAD) biosynthesis, and its activity is critical for the replenishment of NAD level as well as cell survival or death. As one of the most important components in the electron transport chain of complex I in mitochondrion, sustained supply of NAD is essential to the maintenance of energy metabolism both in normal and cancer cells. Recent research shown that X-ray radiation sharply downregulated the expression of NAMPT, which may be the main cause of radiation damage in salivary gland. Consistently, upregulation of NAMPT by phenylephrine restored the function and tissue structure of salivary gland, indicating the cytoprotective role of NAMPT in preventing radiation damage in normal tissues of patients with head and neck cancer during radiotherapy. On the other hand, NAMPT downregulation and NAD depletion could induce cell death in oral squamous cell cancer, suggesting that a combination of NAMPT inhibitor and radiotherapy presents a promising therapeutic strategy for cancer treatment. Based on our and other's studies, NAMPT may have dual roles in cancer radiotherapy: the upregulation of NAMPT could serve to suppress radiotherapy complications such as radiation sialadenitis, and combination regimens that involve NAMPT inhibitors may enhance efficacy of radiotherapy for cancer treatment.
  22. Blood. 2021 Aug 19. pii: blood.2021011010. [Epub ahead of print]
      Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signalling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilisation of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarised mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be of important consideration when looking for treatments of chronic inflammatory conditions where HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.
  23. Commun Biol. 2021 Aug 16. 4(1): 974
      Human ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.
  24. Biochem J. 2021 Aug 16. pii: BCJ20210342. [Epub ahead of print]
      Recent advances in genome sequencing have led to the identification of new ion and metabolite transporters, many of which have not been characterized. Due to the variety of subcellular localizations, cargo and transport mechanisms, such characterization is a daunting task, and predictive approaches focused on the functional context of transporters are very much needed. Here we present a case for identifying a transporter localization using evolutionary rate covariation (ERC), a computational approach based on pairwise correlations of amino acid sequence evolutionary rates across the mammalian phylogeny. As a case study, we find that poorly characterized transporter SLC30A9 (ZnT9) coevolves with several components of the mitochondrial oxidative phosphorylation chain, suggesting mitochondrial localization. We confirmed this computational finding experimentally using recombinant human SLC30A9. SLC30A9 loss caused zinc mishandling in the mitochondria, suggesting that under normal conditions it acts as a zinc exporter. We therefore propose that ERC can be used to predict the functional context of novel transporters and other poorly characterized proteins.
    Keywords:  cellular localization; evolutionary biology; molecular evolution; zinc transport
  25. Free Radic Biol Med. 2021 Aug 14. pii: S0891-5849(21)00471-8. [Epub ahead of print]174 157-170
      Tumor hypoxia is a major biological factor that drives resistance to chemotherapy and radiotherapy. We previously demonstrated that the pro-oxidative drug dihydroartemisinin (DHA) efficiently targeted normoxic and hypoxic cancer cells. Although well studied in normoxia, the mechanism behind DHA-mediated cytotoxicity in hypoxia is insufficiently explored. Here, we analyzed the effect of DHA in HCT116 wild type (wt) cells and in HCT116 Bax-/-Baksh cells with a defective intrinsic apoptosis pathway. Normoxic HCT116 wt cells underwent apoptosis shortly after treatment with DHA. Autophagy-associated cell death contributes to short-term cytotoxicity of DHA in normoxia. These cells switched to an apoptosis- and autophagy-independent cell death after treatment with DHA in hypoxia and displayed similar long-term survival in response to DHA in normoxia and hypoxia. In HCT116 Bax-/-Baksh cells, DHA induced cell cycle arrest shortly after treatment irrespective of oxygen levels. Later, HCT116 Bax-/-Baksh cells induced a delayed cell death after treatment with DHA in hypoxia followed by return to normoxia, while treatment with DHA in normoxia was hardly toxic. We identified lower glutathione levels in hypoxic HCT116 cells which correlated with higher lipid peroxidation after treatment with DHA. Moreover, insufficient expression of Bax/Bak counteracted hypoxia-mediated downregulation of mitochondrial function, thereby adding to DHA-induced ROS production and lipid peroxidation in hypoxia. In summary, DHA-mediated cytotoxicity in normoxia depended on Bax/Bak expression, while cytotoxicity after treatment with DHA in hypoxia was regulated independently of Bax/Bak in HCT116 colorectal cancer cells.
    Keywords:  Apoptosis; Dihydroartemisinin; Glutathione; Hypoxia; Lipid peroxidation; Reactive oxygen species; Therapy resistance
  26. Front Oncol. 2021 ;11 697408
      Under dysbiosis, a gut metabolic disorder, short-chain carboxylic acids (SCCAs) are secreted to the lumen, affecting colorectal cancer (CRC) development. Butyrate and propionate act as CRC growth inhibitors, but they might also serve as carbon source. In turn, the roles of acetate as metabolic fuel and protein acetylation promoter have not been clearly elucidated. To assess whether acetate favors CRC growth through active mitochondrial catabolism, a systematic study evaluating acetate thiokinase (AcK), energy metabolism, cell proliferation, and invasiveness was performed in two CRC cell lines incubated with physiological SCCAs concentrations. In COLO 205, acetate (+glucose) increased the cell density (50%), mitochondrial protein content (3-10 times), 2-OGDH acetylation, and oxidative phosphorylation (OxPhos) flux (36%), whereas glycolysis remained unchanged vs. glucose-cultured cells; the acetate-induced OxPhos activation correlated with a high AcK activity, content, and acetylation (1.5-6-fold). In contrast, acetate showed no effect on HCT116 cell growth, OxPhos, AcK activity, protein content, and acetylation. However, a substantial increment in the HIF-1α content, HIF-1α-glycolytic protein targets (1-2.3 times), and glycolytic flux (64%) was observed. Butyrate and propionate decreased the growth of both CRC cells by impairing OxPhos flux through mitophagy and mitochondrial fragmentation activation. It is described, for the first time, the role of acetate as metabolic fuel for ATP supply in CRC COLO 205 cells to sustain proliferation, aside from its well-known role as protein epigenetic regulator. The level of AcK determined in COLO 205 cells was similar to that found in human CRC biopsies, showing its potential role as metabolic marker.
    Keywords:  acetate thiokinase; acetylation; cancer biomarker; colon cancer; oxidative phoshorylation
  27. ACS Omega. 2021 Aug 10. 6(31): 20676-20685
      Trialkyl phosphonium derivatives of vinyl-substituted p-chlorophenol were synthesized here by a recently developed method of preparing quaternary phosphonium salts from phosphine oxides using Grignard reagents. All the derivatives with a number (n) of carbon atoms in phosphonium alkyl substituents varying from 4 to 7 showed pronounced uncoupling activity in isolated rat liver mitochondria at micromolar concentrations, with a tripentyl derivative being the most effective both in accelerating respiration and causing membrane potential collapse, as well as in provoking mitochondrial swelling in a potassium-acetate medium. Remarkably, the trialkyl phosphonium derivatives with n from 4 to 7 also proved to be rather potent antibacterial agents. Methylation of the chlorophenol hydroxyl group suppressed the effects of P555 and P444 on the respiration and membrane potential of mitochondria but not those of P666, thereby suggesting a mechanistic difference in the mitochondrial uncoupling by these derivatives, which was predominantly protonophoric (carrier-like) in the case of P555 and P444 but detergent-like with P666. The latter was confirmed by the carboxyfluorescein leakage assay on model liposomal membranes.
  28. Sci Rep. 2021 Aug 18. 11(1): 16745
      The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.
  29. Bioorg Chem. 2021 Aug 09. pii: S0045-2068(21)00635-0. [Epub ahead of print]115 105258
      Hsp90 (i.e., Heat shock protein 90) is a well-established therapeutic target for several diseases, ranging from misfolding-related disfunctions to cancer. In this framework, we have developed in recent years a family of benzofuran compounds that act as Hsp90 allosteric modulators. Such molecules can interfere with the stability of some relevant Hsp90 client oncoproteins, showing a low μM cytotoxic activity in vitro in cancer cell lines. Here we identify the target profile of these chemical probes by means of chemical proteomics, which established MDH2 (mitochondrial malate dehydrogenase) as an additional relevant cellular target that might help elucidate the molecular mechanism of their citotoxicity. Western blotting, DARTS (i.e., Drug Affinity Responsive Target Stability) and enzymatic assays data confirmed a dose-dependent interaction of MDH2 with several members of the benzofuran Hsp90 modulators family and a computational model allowed to interpret the observed interactions.
    Keywords:  Benzofurans; Chemical proteomics; Docking; Hsp90; Mitochondrial malate dehydrogenase
  30. Front Cell Dev Biol. 2021 ;9 713729
      Mitochondria are the powerhouses of mammalian cells, which participate in series of metabolic processes and cellular events. Mitochondria have their own genomes, and it is generally acknowledged that human mitochondrial genome encodes 13 proteins, 2 rRNAs and 22 tRNAs. However, the complexity of mitochondria derived transcripts is just starting to be envisaged. Currently, there are at least 8 lncRNAs, some dsRNAs, various small RNAs, and hundreds of circRNAs known to be generated from mitochondrial genome. These non-coding RNAs either translocate into cytosol/nucleus or reside in mitochondria to play various biological functions. Here we present an overview of regulatory non-coding RNAs encoded by the mammalian mitochondria genome. For overall understandings of non-coding RNAs in mitochondrial function, a brief summarization of nuclear-encoded non-coding RNAs in mitochondria is also included. We discuss about roles of these non-coding RNAs in cellular physiology and the communication between mitochondria and the nucleus.
    Keywords:  circRNA; dsRNA; lncRNA; mitochondria; mitochondria-encoded non-coding RNA; small ncRNA
  31. J Biol Chem. 2021 Aug 14. pii: S0021-9258(21)00884-X. [Epub ahead of print] 101081
      The human APOBEC3A (A3A) cytidine deaminase is a powerful DNA mutator enzyme recognized as a major source of somatic mutations in tumor cell genomes. However, there is a discrepancy between APOBEC3A mRNA levels after interferon stimulation in myeloid cells and A3A detection at the protein level. To understand this difference, we investigated the expression of two novel alternative "A3Alt" proteins encoded in the +1-shifted reading frame of the APOBEC3A gene. A3Alt-L and its shorter isoform A3Alt-S appear to be transmembrane proteins targeted to the mitochondrial compartment that induce membrane depolarization and apoptosis. Thus, the APOBEC3A gene represents a new example wherein a single gene encodes two pro-apoptotic proteins, A3A cytidine deaminases that target the genome and A3Alt proteins that target mitochondria.
    Keywords:  A3Alt; APOBEC3A; apoptosis; mitochondria
  32. Small. 2021 Aug 19. e2103086
      Mitochondrial dysfunction is considered to be an important factor that leads to aging and premature aging diseases. Transferring mitochondria to cells is an emerging and promising technique for the therapy of mitochondrial deoxyribonucleic acid (mtDNA)-related diseases. This paper presents a unique method of controlling the quality and quantity of mitochondria transferred to single cells using an automated optical tweezer-based micromanipulation system. The proposed method can automatically, accurately, and efficiently collect and transport healthy mitochondria to cells, and the recipient cells then take up the mitochondria through endocytosis. The results of the study reveal the possibility of using mitochondria from fetal mesenchymal stem cells (fMSCs) as a potential source to reverse the aging-related phenotype and improve metabolic activities in adult mesenchymal stem cells (aMSCs). The results of the quantitative polymerase chain reaction analysis show that the transfer of isolated mitochondria from fMSCs to a single aMSC can significantly increase the antiaging and metabolic gene expression in the aMSC. The proposed mitochondrial transfer method can greatly promote precision medicine for cell therapy of mtDNA-related diseases.
    Keywords:  antiaging; automatic micromanipulation; microfluidics; mitochondrial transfer; optical tweezers
  33. Nature. 2021 Aug 18.
      Fructose consumption is linked to the rising incidence of obesity and cancer, which are two of the leading causes of morbidity and mortality globally1,2. Dietary fructose metabolism begins at the epithelium of the small intestine, where fructose is transported by glucose transporter type 5 (GLUT5; encoded by SLC2A5) and phosphorylated by ketohexokinase to form fructose 1-phosphate, which accumulates to high levels in the cell3,4. Although this pathway has been implicated in obesity and tumour promotion, the exact mechanism that drives these pathologies in the intestine remains unclear. Here we show that dietary fructose improves the survival of intestinal cells and increases intestinal villus length in several mouse models. The increase in villus length expands the surface area of the gut and increases nutrient absorption and adiposity in mice that are fed a high-fat diet. In hypoxic intestinal cells, fructose 1-phosphate inhibits the M2 isoform of pyruvate kinase to promote cell survival5-7. Genetic ablation of ketohexokinase or stimulation of pyruvate kinase prevents villus elongation and abolishes the nutrient absorption and tumour growth that are induced by feeding mice with high-fructose corn syrup. The ability of fructose to promote cell survival through an allosteric metabolite thus provides additional insights into the excess adiposity generated by a Western diet, and a compelling explanation for the promotion of tumour growth by high-fructose corn syrup.
  34. Nat Immunol. 2021 Aug 19.
      Follicular helper T (TFH) cells are a specialized subset of CD4+ T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of TFH cell survival remains unclear. Here we report that TFH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for TFH cell survival. The deletion of GPX4 in T cells selectively abrogated TFH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased TFH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium-GPX4-ferroptosis axis in regulating TFH homeostasis, which can be targeted to enhance TFH cell function in infection and following vaccination.
  35. Nat Commun. 2021 08 17. 12(1): 4841
      RAS proteins are GTPases that lie upstream of a signaling network impacting cell fate determination. How cells integrate RAS activity to balance proliferation and cellular senescence is still incompletely characterized. Here, we identify ZNF768 as a phosphoprotein destabilized upon RAS activation. We report that ZNF768 depletion impairs proliferation and induces senescence by modulating the expression of key cell cycle effectors and established p53 targets. ZNF768 levels decrease in response to replicative-, stress- and oncogene-induced senescence. Interestingly, ZNF768 overexpression contributes to bypass RAS-induced senescence by repressing the p53 pathway. Furthermore, we show that ZNF768 interacts with and represses p53 phosphorylation and activity. Cancer genomics and immunohistochemical analyses reveal that ZNF768 is often amplified and/or overexpressed in tumors, suggesting that cells could use ZNF768 to bypass senescence, sustain proliferation and promote malignant transformation. Thus, we identify ZNF768 as a protein linking oncogenic signaling to the control of cell fate decision and proliferation.
  36. Sci Adv. 2021 Aug;pii: eabh1275. [Epub ahead of print]7(34):
      Genome-wide loss-of-function screens have revealed genes essential for cancer cell proliferation, called cancer dependencies. It remains challenging to link cancer dependencies to the molecular compositions of cancer cells or to unscreened cell lines and further to tumors. Here, we present DeepDEP, a deep learning model that predicts cancer dependencies using integrative genomic profiles. It uses a unique unsupervised pretraining that captures unlabeled tumor genomic representations to improve the learning of cancer dependencies. We demonstrated DeepDEP's improvement over conventional machine learning methods and validated the performance with three independent datasets. By systematic model interpretations, we extended the current dependency maps with functional characterizations of dependencies and a proof-of-concept in silico assay of synthetic essentiality. We applied DeepDEP to pan-cancer tumor genomics and built the first pan-cancer synthetic dependency map of 8000 tumors with clinical relevance. In summary, DeepDEP is a novel tool for investigating cancer dependency with rapidly growing genomic resources.
  37. STAR Protoc. 2021 Sep 17. 2(3): 100721
      Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted imaging processing approach. For complete details on the use and execution of this protocol, please refer to Miyamoto et al., 2021.
    Keywords:  Biotechnology and bioengineering; Cell Biology; Molecular/Chemical Probes; Protein Biochemistry
  38. Hum Mol Genet. 2021 Aug 20. pii: ddab240. [Epub ahead of print]
    NHLBI Trans-Omics for Precision Medicine (TOPMed) Consortium
      We conducted cohort- and race-specific epigenome-wide association analyses of mtDNA copy number (mtDNA CN) measured in whole blood from participants of African and European origins in five cohorts (n = 6182, mean age 57-67 years, 65% women). In the meta-analysis of all the participants, we discovered 21 mtDNA CN-associated CpG sites (p < 1 x 10-7), with a 0.7 to 3.0 standard deviation increase (3 CpGs) or decrease (18 CpGs) in mtDNA CN corresponding to a 1% increase in DNA methylation. Several significant CpGs have been reported to be associated with at least two risk factors (e.g. chronological age or smoking) for cardiovascular disease (CVD). Five genes (PRDM16, NR1H3, XRCC3, POLK, and PDSS2), which harbor nine significant CpGs, are known to be involved in mitochondrial biosynthesis and functions. For example, NR1H3 encodes a transcription factor that is differentially expressed during an adipose tissue transition. The methylation level of cg09548275 in NR1H3 was negatively associated with mtDNA CN (effect size = -1.71, p = 4 x 10-8) and positively associated with the NR1H3 expression level (effect size = 0.43, p = 0.0003), which indicates that the methylation level in NR1H3 may underlie the relationship between mtDNA CN, the NR1H3 transcription factor, and energy expenditure. In summary, the study results suggest that mtDNA CN variation in whole blood is associated with DNA methylation levels in genes that are involved in a wide range of mitochondrial activities. These findings will help reveal molecular mechanisms between mtDNA CN and CVD.
  39. STAR Protoc. 2021 Sep 17. 2(3): 100687
      Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity. For complete details on the use and execution of this protocol, please refer to Divakaruni et al. (2018).
    Keywords:  Cell separation/fractionation; Cell-based Assays; Metabolism
  40. J Food Biochem. 2021 Aug 16. e13895
      Avocatin-B (Avo-B), an avocado-derived 1:1 mixture of the polyhydroxylated alcohols avocadyne (AYNE) and avocadene, eliminated leukemia cells by suppressing fatty acid oxidation (FAO) in vivo and in vitro while sparing healthy blood cells. In this study, we identified AYNE as the most potent FAO inhibitor within the Avo-B mixture capable of inducing cell death in leukemia cells lines (IC50 : 3.10 ± 0.14 µM in TEX cells; 11.53 ± 3.32 µM in OCI-AML2) and patient-derived acute myeloid leukemia cells. When added individually, the two Avo-B constituents demonstrated antagonism (Combination Index values >1), highlighting the need for future studies to assess AYNE alone. Together, this work highlighted AYNE as the most potent FAO inhibitor within the Avo-B mixture. PRACTICAL APPLICATIONS: This work identifies which of the two molecules in avocatin B (Avo-B), an avocado-derived mixture of two molecules with demonstrated human safety, utility against leukemia, insulin resistance and diabetes, is most useful. Therefore, it provides the basis for future clinical studies that will focus on testing and developing the most active Avo-B constituent.
    Keywords:  avocado; cancer; fatty acid oxidation
  41. FEBS J. 2021 Aug 17.
      Accumulation of mutations such as deletions in mitochondrial DNA is associated with ageing, cancer and human genetic disorders. These deletions are often flanked by GC-skewed sequence motifs that can potentially fold into secondary non-B DNA conformations. G-quadruplexes are emerging as key initiators of mitochondrial genomic instability. In this issue, Dahal et al provide an in silico analysis of sequence motifs that can fold into altered DNA structures in mitochondrial genomic regions that contain frequent deletions. They show the formation of five G-quadruplexes near such frequent breakpoints using biochemical and biophysical approaches in vitro and more importantly inside mammalian cells. Comment on:
    Keywords:  G4; Non-B DNA; genomic instability; mitochondrial DNA deletions
  42. Bioorg Chem. 2021 Aug 08. pii: S0045-2068(21)00626-X. [Epub ahead of print]115 105249
      A large number of derivatives of natural pentacyclic triterpenoid oleanolic acid (OA) with various activities have been reported, including CDDO derivatives (CDDOs). CDDOs show potent antitumor activity, but they lack selectivity for tumor cells which causes serious side effects. In this study, based on the truth that tumor cells display higher mitochondrial membrane potential, to improve their mitochondrial-targeting ability, triphenylphosphine cations (TPP+) or tricyclohexylphosphine cations (TCP+) were linked to CDDO. Among these compounds, the TPP+ derivative 5b exhibited greater activity against the tumor cells than CDDO-Me, and the selectivity for the tumor cells was obviously improved. Further investigation revealed that the uptake of 5b in the mitochondria of MCF-7 cells was increased compared to CDDO-Me. In addition, 5b was able to cause mitochondrial membrane potential decline and cell cycle arrest. Furthermore, 5b caused apoptosis mainly through the mitochondria-mediated intrinsic pathway. Taken together, our study provides a possible solution to the poor selectivity of CDDOs, and regains confidence in the treatment of tumor with CDDOs.
    Keywords:  Antitumor; CDDO derivatives; Cell cycle arrest; Mitochondrial-targeting
  43. Cancer Res. 2021 Aug 16. pii: canres.CAN-21-0757-A.2021. [Epub ahead of print]
      The most recent American Dietary Guidelines (2020-2025) recommend shifting dietary fats from solid saturated fats to unsaturated oils. Dietary oils contain different compositions of unsaturated fatty acids (UFA). Oleic acid (OA) and linoleic acid (LA) are the most common UFA in dietary oils. How individual UFA in oils regulate immune cell function and cancer risk remains unclear. Here we demonstrated that high fat diets (HFD) rich either in OA or LA induced a similar degree of murine obesity, but the LA-rich HFD specifically promoted mammary tumor growth. LA impaired anti-tumor T cell responses by promoting naïve T cell apoptosis and inhibiting TNFα production. While exogenous OA and LA were taken up by T cells with similar efficacy, only LA induced significant mitochondrial ROS production and lipid peroxidation. Importantly, naïve T cells predominantly expressed epidermal fatty acid binding protein (E-FABP), which is central in facilitating LA mitochondrial transport and cardiolipin incorporation. Genetic depletion of E-FABP rescued LA-impaired T cell responses and suppressed LA-rich HFD-associated mammary tumor growth. Collectively, these data suggest that dietary oils high in LA promote mammary tumors by inducing E-FABP-mediated T cell dysfunction.
  44. Nat Cancer. 2020 Jun;1(6): 589-602
      Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis1-3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.
  45. Cancer Metab. 2021 Aug 16. 9(1): 31
      BACKGROUND: Metabolic reprogramming is a central feature in many cancer subtypes and a hallmark of cancer. Many therapeutic strategies attempt to exploit this feature, often having unintended side effects on normal metabolic programs and limited efficacy due to integrative nature of metabolic substrate sourcing. Although the initiating oncogenic lesion may vary, tumor cells in lymphoid malignancies often share similar environments and potentially similar metabolic profiles. We examined cells from mouse models of MYC-, RAS-, and BCR-ABL-driven lymphoid malignancies and find a convergence on de novo lipogenesis. We explore the potential role of MYC in mediating lipogenesis by 13C glucose tracing and untargeted metabolic profiling. Inhibition of lipogenesis leads to cell death both in vitro and in vivo and does not induce cell death of normal splenocytes.METHODS: We analyzed RNA-seq data sets for common metabolic convergence in lymphoma and leukemia. Using in vitro cell lines derived in from conditional MYC, RAS, and BCR-ABL transgenic murine models and oncogene-driven human cell lines, we determined gene regulation, metabolic profiles, and sensitivity to inhibition of lipogenesis in lymphoid malignancies. We utilize preclinical murine models and transgenic primary model of T-ALL to determine the effect of lipogenesis blockade across BCR-ABL-, RAS-, and c-MYC-driven lymphoid malignancies. Statistical significance was calculated using unpaired t-tests and one-way ANOVA.
    RESULTS: This study illustrates that de novo lipid biogenesis is a shared feature of several lymphoma subtypes. Using cell lines derived from conditional MYC, RAS, and BCR-ABL transgenic murine models, we demonstrate shared responses to inhibition of lipogenesis by the acetyl-coA carboxylase inhibitor 5-(tetradecloxy)-2-furic acid (TOFA), and other lipogenesis inhibitors. We performed metabolic tracing studies to confirm the influence of c-MYC and TOFA on lipogenesis. We identify specific cell death responses to TOFA in vitro and in vivo and demonstrate delayed engraftment and progression in vivo in transplanted lymphoma cell lines. We also observe delayed progression of T-ALL in a primary transgenic mouse model upon TOFA administration. In a panel of human cell lines, we demonstrate sensitivity to TOFA treatment as a metabolic liability due to the general convergence on de novo lipogenesis in lymphoid malignancies driven by MYC, RAS, or BCR-ABL. Importantly, cell death was not significantly observed in non-malignant cells in vivo.
    CONCLUSIONS: These studies suggest that de novo lipogenesis may be a common survival strategy for many lymphoid malignancies and may be a clinically exploitable metabolic liability.
    TRIAL REGISTRATION: This study does not include any clinical interventions on human subjects.
    Keywords:  ACACA; BCR-ABL; Cancer metabolism; De novo lipogenesis; FASN; Fatty acid synthesis; Lipogenesis; Lymphoma; Oncogene addiction; RAS; T-ALL; c-MYC