bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒10‒17
forty-five papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Cancer Metab. 2021 Oct 09. 9(1): 36
      BACKGROUND: Enhanced metabolic plasticity and diversification of energy production is a hallmark of highly proliferative breast cancers. This contributes to poor pharmacotherapy efficacy, recurrence, and metastases. We have previously identified a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively reduces bioenergetic coupling efficiency and is orally available. Here, we evaluated the antineoplastic properties of uncoupling oxidative phosphorylation from ATP production in breast cancer using BAM15.METHODS: The anticancer effects of BAM15 were evaluated in human triple-negative MDA-MB-231 and murine luminal B, ERα-negative EO771 cells as well as in an orthotopic allograft model of highly proliferative mammary cancer in mice fed a standard or high fat diet (HFD). Untargeted transcriptomic profiling of MDA-MB-231 cells was conducted after 16-h exposure to BAM15. Additionally, oxidative phosphorylation and electron transfer capacity was determined in permeabilized cells and excised tumor homogenates after treatment with BAM15.
    RESULTS: BAM15 increased proton leak and over time, diminished cell proliferation, migration, and ATP production in both MDA-MB-231 and EO771 cells. Additionally, BAM15 decreased mitochondrial membrane potential, while inducing apoptosis and reactive oxygen species accumulation in MDA-MB-231 and EO771 cells. Untargeted transcriptomic profiling of MDA-MB-231 cells further revealed inhibition of signatures associated with cell survival and energy production by BAM15. In lean mice, BAM15 lowered body weight independent of food intake and slowed tumor progression compared to vehicle-treated controls. In HFD mice, BAM15 reduced tumor growth relative to vehicle and calorie-restricted weight-matched controls mediated in part by impaired cell proliferation, mitochondrial respiratory function, and ATP production. LC-MS/MS profiling of plasma and tissues from BAM15-treated animals revealed distribution of BAM15 in adipose, liver, and tumor tissue with low abundance in skeletal muscle.
    CONCLUSIONS: Collectively, these data indicate that mitochondrial uncoupling may be an effective strategy to limit proliferation of aggressive forms of breast cancer. More broadly, these findings highlight the metabolic vulnerabilities of highly proliferative breast cancers which may be leveraged in overcoming poor responsiveness to existing therapies.
    Keywords:  BAM15; Breast cancer; Cell proliferation; Mitochondrial function; Tumor metabolism
  2. Cell Death Discov. 2021 Oct 14. 7(1): 293
      Liver cancer is one of the most common and lethal types of oncological disease in the world, with limited treatment options. New treatment modalities are desperately needed, but their development is hampered by a lack of insight into the underlying molecular mechanisms of disease. It is clear that metabolic reprogramming in mitochondrial function is intimately linked to the liver cancer process, prompting the possibility to explore mitochondrial biochemistry as a potential therapeutic target. Here we report that depletion of mitochondrial DNA, pharmacologic inhibition of mitochondrial electron transport chain (mETC) complex I/complex III, or genetic of mETC complex I restricts cancer cell growth and clonogenicity in various preclinical models of liver cancer, including cell lines, mouse liver organoids, and murine xenografts. The restriction is linked to the production of reactive oxygen species, apoptosis induction and reduced ATP generation. As a result, our findings suggest that the mETC compartment of mitochondria could be a potential therapeutic target in liver cancer.
  3. Nat Commun. 2021 Oct 13. 12(1): 5977
      Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.
  4. Mol Cancer Res. 2021 Oct 11. pii: molcanres.0385.2021. [Epub ahead of print]
      Lung adenocarcinoma and squamous cell carcinoma are two most common subtypes of lung cancer. Here, to identify new, targetable molecular properties of both subtypes, we monitored changes in the levels of heme- and oxidative phosphorylation (OXPHOS)-related proteins during lung tumorigenesis. Heme is a central molecule for oxidative metabolism and ATP generation via OXPHOS. Notably, both lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors can be induced in the genetically engineered KLLuc mouse model harboring the G12D Kras mutation and a conditional Lkb1 knockout. We found that the levels of the rate-limiting heme synthesis enzyme ALAS1 and uptake protein SLC48A1, along with oxidative phosphorylation (OXPHOS) complex subunits, progressively increased as lung tumorigenesis advanced. Our data demonstrated that elevated levels of heme- and OXPHOS-related proteins were associated with both ADC and SCC. Importantly, treatment of KLLuc mice with a heme-sequestering protein HeSP2 that inhibits heme uptake in tumor cells effectively arrested lung tumor progression, and both ADC and SCC tumors were strongly suppressed. Additionally, HeSP2 effectively suppressed the growth of both SCC and ADC tumor xenografts in NOD/SCID mice. Further analyses indicated that HeSP2 effectively diminished OXPHOS in both ADC and SCC, reduced angiogenesis, alleviated tumor hypoxia, and suppressed cell proliferation. These results show that the advancing of lung tumorigenesis requires progressive increase in cellular heme synthesis and uptake, leading to intensified OXPHOS activity and ATP generation and promoting aggressive tumorigenic functions. Implications: Heme sequestration is an effective strategy for the suppression of both ADC and SCC tumor initiation and development.
  5. STAR Protoc. 2021 Dec 17. 2(4): 100850
      This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020).
    Keywords:  Biotechnology and bioengineering; Cell Biology; Cell culture; Cell-based Assays; Metabolism
  6. Chem Biol Interact. 2021 Oct 08. pii: S0009-2797(21)00315-X. [Epub ahead of print] 109677
      Methylglyoxal (MG) is a reactive metabolite derived from different physiological pathways. Its production can be harmful to cells via glycation reactions of lipids, DNA, and proteins. But, the effects of MG on mitochondrial functioning and bioenergetic responses are still elusive. Then, the effects of MG on key parameters of mitochondrial functionality were examined here. Isolated rat liver mitochondria were exposed to 0.1-10 mM of MG to determine its toxicity in the mitochondrial viability, membrane potential (Δψm), swelling and the superoxide (O2•-) production. Besides, mitochondrial oxidative phosphorylation parameters were analyzed by high-resolution respiratory (HRR) assay. In this set of experiments, routine state, PM state (pyruvate/malate), oxidative phosphorylation (OXPHOS), LEAK respiration, electron transport system (ETS) and oxygen residual (ROX) states were evaluated. HRR showed that PM state, OXPHOS CI-Linked, LEAK respiration, ETS CI/CII-Linked and ETS CII-Linked/ROX were significantly inhibited by MG exposure. MG also inhibited the complex II activity, and decreased Δψm and the viability of mitochondria. Taken together, our data indicates that MG is an inductor of mitochondrial dysfunctions and impairs important steps of respiratory chain, effects that can alter bioenergetics responses.
    Keywords:  High-resolution respirometry; Methylglyoxal; Mitochondria; Oxidative stress
  7. Molecules. 2021 Sep 26. pii: 5842. [Epub ahead of print]26(19):
      Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.
    Keywords:  Bixafen; Boscalid; EPR; SDHI; mitochondria; oxygen consumption rate (OCR); superoxide
  8. Cancers (Basel). 2021 Oct 05. pii: 4994. [Epub ahead of print]13(19):
      miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.
    Keywords:  FOXJ3; colorectal cancer; miRNA; mitochondria; tumor metabolism
  9. Mol Metab. 2021 Oct 09. pii: S2212-8778(21)00201-5. [Epub ahead of print] 101354
      OBJECTIVE: ATM, the protein defective in the human genetic disorder, ataxia telangiectasia (A-T) plays a central role in the response to DNA double strand breaks (DSBs) and in protecting the cell against oxidative stress. We recently showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress.METHODS: We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2 deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 hours using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS to determine altered levels of TCA intermediates A-T cells compared to healthy control cells.
    RESULTS: We demonstrate here that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate.
    CONCLUSIONS: These data together show that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has great potential as a novel therapeutic agent for patients with A-T.
    Keywords:  ATM; Ataxia-telangiectasia; endoplasmic reticulum; heptanoate (C7); mitochondrial dysfunction; mitochondrial interaction; nutrient deprivation
  10. iScience. 2021 Oct 22. 24(10): 103153
      The dual function protein ACAD9 catalyzes α,β-dehydrogenation of fatty acyl-CoA thioesters in fatty acid β-oxidation and is an essential chaperone for mitochondrial respiratory complex I (CI) assembly. ACAD9, ECSIT, and NDUFAF1 interact to form the core mitochondrial CI assembly complex. Current studies examine the molecular mechanism of ACAD9/ECSIT/NDUFAF1interactions. ACAD9 binds to the carboxy-terminal half and NDUFAF1 to the amino-terminal half of ECSIT. Binary complexes are unstable and aggregate easily, while the ACAD9/ECSIT/NDUFAF1 ternary complex is soluble and highly stable. Molecular modeling and small-angle X-ray scattering studies identified intra-complex interaction sites and binding sites for other assembly factors. Binding of ECSIT at the ETF binding site in the amino-terminal domain of ACAD9 is consistent with observed loss of FAD and enzymatic activity and demonstrates that the two functions of ACAD9 are mutually exclusive. Mapping of 42 known pathogenic mutations onto the homology-modeled ACAD9 structure provides structural insights into pathomechanisms of CI deficiency.
    Keywords:  Biological sciences; Molecular biology; Structural biology
  11. Int J Mol Sci. 2021 Oct 06. pii: 10790. [Epub ahead of print]22(19):
      Metabolic transformation of cancer cells leads to the accumulation of lactate and significant acidification in the tumor microenvironment. Both lactate and acidosis have a well-documented impact on cancer progression and negative patient prognosis. Here, we report that cancer cells adapted to acidosis are significantly more sensitive to oxidative damage induced by hydrogen peroxide, high-dose ascorbate, and photodynamic therapy. Higher lactate concentrations abrogate the sensitization. Mechanistically, acidosis leads to a drop in antioxidant capacity caused by a compromised supply of nicotinamide adenine dinucleotide phosphate (NADPH) derived from glucose metabolism. However, lactate metabolism in the Krebs cycle restores NADPH supply and antioxidant capacity. CPI-613 (devimistat), an anticancer drug candidate, selectively eradicates the cells adapted to acidosis through inhibition of the Krebs cycle and induction of oxidative stress while completely abrogating the protective effect of lactate. Simultaneous cell treatment with tetracycline, an inhibitor of the mitochondrial proteosynthesis, further enhances the cytotoxic effect of CPI-613 under acidosis and in tumor spheroids. While there have been numerous attempts to treat cancer by neutralizing the pH of the tumor microenvironment, we alternatively suggest considering tumor acidosis as the Achilles' heel of cancer as it enables selective therapeutic induction of lethal oxidative stress.
    Keywords:  CPI-613; acidosis; bioenergetics; cancer; lactate; mitochondria; photodynamic therapy; tetracycline; therapy; tumor microenvironment
  12. Cancer Sci. 2021 Oct 10.
      Ovarian carcinoma immune-reactive antigen domain 2 (OCIAD2) has been reported to show significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, and its abnormal expression is associated with poorer prognosis of the patients. However, the cellular function of OCIAD2 in this tumor remains poorly understood. In the present study, we first validated that OCIAD2 showed higher expression in human lung adenocarcinoma tissues or cell lines than in normal lung tissue or immortalized normal bronchial epithelial cells. OCIAD2 was localized predominantly at the mitochondrial membrane in lung adenocarcinoma cells. Interestingly, suppression of OCIAD2 led to loss of mitochondrial structure and a reduction in the number of mitochondria. Moreover, OCIAD2 suppression led to down-regulation of cellular growth, proliferation, migration and invasion, and up-regulation of mitochondria-related apoptosis. We also showed that OCIAD2 suppression induced a decrease of mitochondrial membrane potential and release of cytochrome c. Transcriptional profiling using RNA sequencing revealed a total of 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827 and PC9). Pathway enrichment analysis of those genes demonstrated significant enrichment in apoptotic signaling or ER stress pathways. Our data suggest that OCIAD2 inhibits the mitochondria-initiating apoptosis and thus promote the survival of lung cancer cells. Therefore, OCIAD2 may be an effective target for treatment of lung adenocarcinoma.
    Keywords:  Electron microscopy; OCIAD2; apoptosis; lung adenocarcinoma; mitochondria
  13. Int J Mol Sci. 2021 Oct 01. pii: 10665. [Epub ahead of print]22(19):
      Breast cancer development is associated with macrophage infiltration and differentiation in the tumor microenvironment. Our previous study highlights the crucial function of reactive oxygen species (ROS) in enhancing macrophage infiltration during the disruption of mammary tissue polarity. However, the regulation of ROS and ROS-associated macrophage infiltration in breast cancer has not been fully determined. Previous studies identified retinoid orphan nuclear receptor alpha (RORα) as a potential tumor suppressor in human breast cancer. In the present study, we showed that retinoid orphan nuclear receptor alpha (RORα) significantly decreased ROS levels and inhibited ROS-mediated cytokine expression in breast cancer cells. RORα expression in mammary epithelial cells inhibited macrophage infiltration by repressing ROS generation in the co-culture assay. Using gene co-expression and chromatin immunoprecipitation (ChIP) analyses, we identified complex I subunits NDUFS6 and NDUFA11 as RORα targets that mediated its function in suppressing superoxide generation in mitochondria. Notably, the expression of RORα in 4T1 cells significantly inhibited cancer metastasis, reduced macrophage accumulation, and enhanced M1-like macrophage differentiation in tumor tissue. In addition, reduced RORα expression in breast cancer tissue was associated with an increased incidence of cancer metastasis. These results provide additional insights into cancer-associated inflammation, and identify RORα as a potential target to suppress ROS-induced mammary tumor progression.
    Keywords:  breast cancer; complex I; macrophage; orphan nuclear receptor; reactive oxygen species; tumor microenvironment
  14. Nat Commun. 2021 Oct 13. 12(1): 5989
      Liquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.
  15. Nat Metab. 2021 Oct 14.
      During early mammalian embryogenesis, changes in cell growth and proliferation depend on strict genetic and metabolic instructions. However, our understanding of metabolic reprogramming and its influence on epigenetic regulation in early embryo development remains elusive. Here we show a comprehensive metabolomics profiling of key stages in mouse early development and the two-cell and blastocyst embryos, and we reconstructed the metabolic landscape through the transition from totipotency to pluripotency. Our integrated metabolomics and transcriptomics analysis shows that while two-cell embryos favour methionine, polyamine and glutathione metabolism and stay in a more reductive state, blastocyst embryos have higher metabolites related to the mitochondrial tricarboxylic acid cycle, and present a more oxidative state. Moreover, we identify a reciprocal relationship between α-ketoglutarate (α-KG) and the competitive inhibitor of α-KG-dependent dioxygenases, L-2-hydroxyglutarate (L-2-HG), where two-cell embryos inherited from oocytes and one-cell zygotes display higher L-2-HG, whereas blastocysts show higher α-KG. Lastly, increasing 2-HG availability impedes erasure of global histone methylation markers after fertilization. Together, our data demonstrate dynamic and interconnected metabolic, transcriptional and epigenetic network remodelling during early mouse embryo development.
  16. J Vis Exp. 2021 Sep 24.
      Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.
  17. Toxicology. 2021 Oct 07. pii: S0300-483X(21)00307-3. [Epub ahead of print] 152985
      Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM, 24 hours) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.
    Keywords:  Phthalates; mitochondria; steroidogenesis
  18. Arch Toxicol. 2021 Oct 13.
      Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.
    Keywords:  DILI; ETC complex inhibitors; High-content imaging; Mitochondrial toxicity; TempO-Seq
  19. Mol Cancer Res. 2021 Oct 15. pii: molcanres.0448.2021. [Epub ahead of print]
      Loss of function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for LKB1-deficient non-small cell lung cancer (NSCLC) patients remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analogue. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the anti-tumor efficacy of 8-Cl-Ado. Implications: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.
  20. PLoS One. 2021 ;16(10): e0256619
      Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.
  21. Proc Jpn Acad Ser B Phys Biol Sci. 2021 ;97(8): 479-498
      Polyphosphate [poly(P)] is described as a homopolymer of inorganic phosphates. Nicotinamide adenine dinucleotide kinase (NAD kinase) catalyzes the phosphorylation of NAD+ to NADP+ in the presence of ATP (ATP-NAD kinase). Novel NAD kinase that explicitly phosphorylates NAD+ to NADP+ using poly(P), besides ATP [ATP/poly(P)-NAD kinase], was found in bacteria, in particular, Gram-positive bacteria, and the gene encoding ATP/poly(P)-NAD kinase was also newly identified in Mycobacterium tuberculosis H37Rv. Both NAD kinases required multi-homopolymeric structures for activity expression. The enzymatic and genetic results, combined with their primary and tertiary structures, have led to the discovery of a long-awaited human mitochondrial NAD kinase. This discovery showed that the NAD kinase is a bacterial type of ATP/poly(P)-NAD kinase. These pioneering findings, i.e., ATP/poly(P)-NAD kinase, NAD kinase gene, and human mitochondrial NAD kinase, have significantly enhanced research on the biochemistry, molecular biology, and evolutionary biology of NAD kinase, mitochondria, and poly(P), including some biotechnological knowledge applicable to NADP+ production.
    Keywords:  NAD kinase; NAD kinase gene; NAD+; NADP+; human mitochondrial NAD kinase; polyphosphate
  22. Int J Mol Sci. 2021 Sep 30. pii: 10600. [Epub ahead of print]22(19):
      Metabolic adaptation to increased oxidative phosphorylation (OXPHOS) has been found in gastrointestinal stromal tumor (GIST) upon imatinib treatment. However, the underlying mechanism of imatinib-induced OXPHOS is unknown. Discovering molecules that mediate imatinib-induced OXPHOS may lead to the development of therapeutic strategies synergizing the efficacy of imatinib. In this study, we explored the role of microRNAs in regulating OXPHOS in GIST upon imatinib treatment. Using a microarray approach, we found that miR-483-3p was one of the most downregulated miRNAs in imatinib-treated tumors compared to untreated tumors. Using an extended series of GIST samples, we further validated the downregulation of miR-483-3p in imatinib-treated GIST samples by RT-qPCR. Using both gain- and loss-of-function experiments, we showed that miR-483-3p could regulate mitochondrial respiratory Complex II expression, suggesting its role in OXPHOS regulation. Functionally, miR-483-3p overexpression could rescue imatinib-induced cell death. These findings provide the molecular link for imatinib-induced OXPHOS expression and the biological role of miR-483-3p in regulating cell viability upon imatinib treatment.
    Keywords:  Complex II; gastrointestinal stromal tumor (GIST); imatinib; miR-483; microRNA; oxidative phosphorylation; succinate dehydrogenase B
  23. Mol Oncol. 2021 Oct 10.
      Multiple molecular features, such as activation of specific oncogenes (e.g. MYC, BCL2) or a variety of gene expression signatures, have been associated with disease course in diffuse large B-cell lymphoma (DLBCL), although their relationships and implications for targeted therapy remain to be fully unraveled. We report that MYC activity is closely correlated with - and most likely a driver of - gene signatures related to oxidative phosphorylation (OxPhos) in DLBCL, pointing to OxPhos enzymes, in particular mitochondrial electron transport chain (ETC) complexes, as possible therapeutic targets in high-grade MYC-associated lymphomas. In our experiments, indeed, MYC sensitized B-cells to the ETC complex I inhibitor IACS-010759. Mechanistically, IACS-010759 triggered the integrated stress response (ISR) pathway, driven by the transcription factors ATF-4 and CHOP, which engaged the intrinsic apoptosis pathway and lowered the apoptotic threshold in MYC-overexpressing cells. In line with these findings, the BCL2-inhibitory compound venetoclax synergized with IACS-010759 against double-hit lymphoma (DHL), a high-grade malignancy with concurrent activation of MYC and BCL2. In BCL2-negative lymphoma cells, instead, killing by IACS-010759 was potentiated by the Mcl-1 inhibitor S63845. Thus, combining an OxPhos inhibitor with select BH3-mimetic drugs provides a novel therapeutic principle against aggressive, MYC-associated DLBCL variants.
    Keywords:  BCL2; DLBCL; Integrated Stress Response; MYC; OxPhos; chemotherapy
  24. Theranostics. 2021 ;11(19): 9470-9491
      Introduction: An imbalance in redox homeostasis consistently inhibits tumor cell proliferation and further causes tumor regression. Thus, synchronous glutaminolysis inhibition and intracellular reactive oxygen (ROS) accumulation cause severe redox dyshomeostasis, which may potentially become a new therapeutic strategy to effectively combat cancer. Methods: Mitochondrial-targeting liposomal nanoparticles (abbreviated MLipRIR NPs) are synthesized by the encapsulation of R162 (inhibitor of glutamate dehydrogenase 1 [GDH1]) and IR780 (a hydrophobic sonosensitizer) within the lipid bilayer, which are exploited for ultrasound (US)-activated tumor dyshomeostasis therapy reinforced by immunogenic cell death (ICD). Results: R162 released from MLipRIR NPs disrupts the glutaminolysis pathway in mitochondria, resulting in downregulated enzymatic activity of glutathione peroxidase (GPx). In addition, loaded IR780 can generate high levels of ROS under US irradiation, which not only interrupts mitochondrial respiration to induce apoptosis but also consumes local glutathione (GSH). GSH depletion accompanied by GPx deactivation causes severe ferroptosis of tumor cells through the accumulation of lipid peroxides. Such intracellular redox dyshomeostasis effectively triggers immunogenic cell death (ICD), which can activate antitumor immunity for the suppression of both primary and distant tumors with the aid of immune checkpoint blockade. Conclusions: Taking advantage of multimodal imaging for therapy guidance, this nanoplatform may potentiate systemic tumor eradication with high certainty. Taken together, this state-of-the-art paradigm may provide useful insights for cancer management by disrupting redox homeostasis.
    Keywords:  drug delivery; liposome; redox dyshomeostasis; sonodynamic therapy; targeting
  25. Mol Cell Endocrinol. 2021 Oct 09. pii: S0303-7207(21)00328-2. [Epub ahead of print] 111484
      Pheochromocytomas (PCCs) are rare neuroendocrine tumors derived from adrenal medulla chromaffin cells. Malignancy and recurrence are rare but demand effective treatment. Metformin exerts antiproliferative effects in several cancer cell lines. We thus evaluated the effects of metformin on cell viability and proliferation, cellular respiration and AMPK-AKT-mTOR-HIFA proliferation pathway on a rat PCC cell line (PC12-Adh). We then addressed metformin's effects on the AMPK-AKT-mTOR-HIFA pathway on two human primary cultures: one from a VHL-mutant PCC and other from a sporadic PCC. Metformin (20 mM) inhibited PC12-Adh cell proliferation, and decreased oxygen consumption, ATP production and proton leak, in addition to loss of mitochondrial membrane potential. Further, metformin induced AMPK phosphorylation and impaired AMPK-PI3k-AKT-mTOR pathway activation. The mTOR pathway was also inhibited in human VHL-related PCC cells, however, in an AMPK-independent manner. Metformin-induced decrease of HIF1A levels was likely mediated by proteasomal degradation. Altogether our results suggest that metformin impairs PCC cellular proliferation.
    Keywords:  Cellular respiration; Metformin; Mitochondria; Pheochromocytoma; Primary culture; VHL
  26. Int J Mol Sci. 2021 Sep 29. pii: 10539. [Epub ahead of print]22(19):
      Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.
    Keywords:  azoxymethane; cationic amphiphilic drugs; colorectal cancer; dextrane sulfate; glucosylceramide synthase; glycosphingolipids
  27. Front Cell Dev Biol. 2021 ;9 737304
      Mitophagy specifically recognizes and removes damaged or superfluous mitochondria to maintain mitochondrial homeostasis and proper neuronal function. Defective mitophagy and the resulting accumulation of damaged mitochondria occur in several neurodegenerative diseases. Previously, we showed mitochondrial dysfunction in astrocytes with POLG mutations, and here, we examined how POLG mutations affect mitophagy in astrocytes and how this can be ameliorated pharmacologically. Using induced pluripotent stem cell (iPSC)-derived astrocytes carrying POLG mutations, we found downregulation of mitophagy/autophagy-related genes using RNA sequencing-based KEGG metabolic pathway analysis. We confirmed a deficit in mitochondrial autophagosome formation under exogenous stress conditions and downregulation of the mitophagy receptor p62, reduced lipidation of LC3B-II, and decreased expression of lysosome protein lysosomal-associated membrane protein 2A (LAMP2A). These changes were regulated by the PINK1/Parkin pathway and AKT/mTOR/AMPK/ULK1 signaling pathways. Importantly, we found that double treatment with nicotinamide riboside (NR) and metformin rescued mitophagy defects and mitochondrial dysfunction in POLG-mutant astrocytes. Our findings reveal that impaired mitophagy is involved in the observed mitochondrial dysfunction caused by POLG mutations in astrocytes, potentially contributing to the phenotype in POLG-related diseases. This study also demonstrates the therapeutic potential of NR and metformin in these incurable mitochondrial diseases.
    Keywords:  IPSC (induced pluripotent stem cells); POLG; astrocytes; metformin; mitochondria; mitophagy; nicotinamide riboside (NR)
  28. Free Radic Biol Med. 2021 Oct 08. pii: S0891-5849(21)00754-1. [Epub ahead of print]
      Whether from known or unknown causes, loss of epithelial repair plays a central role in the pathogenesis of pulmonary fibrosis. Recently, diminished mitochondrial function has been implicated as a factor contributing to the loss of epithelial repair but the mechanisms mediating these changes have not been defined. Here, we investigated the factors contributing to mitochondrial respiratory dysfunction after bleomycin, a widely accepted agent for modeling pulmonary fibrosis in mice and in vitro systems. In agreement with previous reports, we found that mitochondrial respiration was decreased in lung epithelial cells exposed to bleomycin, but we also observed that responses differed depending on the type of metabolic fuel available to cells. For example, we found that mitochondrial respiration was dramatically reduced when glucose served as the primary fuel. Moreover, this associated with a marked decrease in glucose uptake, expression of glucose uptake transport 1 and capacity to augment glycolysis to either glucose or oligomycin. Conversely, mitochondrial respiration was largely preserved if glutamine was present in culture medium. The addition of glutamine also lead to increased intracellular metabolite levels, including multiple TCA cycle intermediates and the glycolytic intermediate lactate, and was associated with reduced DNA damage and cell death to bleomycin. In summary, our findings indicate that glutamine, rather than glucose, supports mitochondrial respiration and metabolite production in injured lung epithelium, and suggest that this shift away from glucose utilization serves to protect the lung epithelium from bleomycin injury.
    Keywords:  13C fluxomics; 13CNMR; DNA damage; Epithelium; Glutamine; LC-MS/MS; Metabolic reprogramming; Mitochondria; Targeted metabolomics
  29. Proc Natl Acad Sci U S A. 2021 Oct 19. pii: e2103803118. [Epub ahead of print]118(42):
      Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I-expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I-overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2-an enzyme without a counterpart in mammals-as a candidate target for leishmanicidal drugs.
    Keywords:   Leishmania ; NADH oxidation; complex I; mitochondria; type II NADH dehydrogenase
  30. J Cell Sci. 2021 Oct 15. pii: jcs.259188. [Epub ahead of print]
      Mutation in isocitrate dehydrogenase 1 (IDH1) gene, leading to the production of oncometabolite D-2-hydroxyglutarate (2-HG) from α-ketoglutarate, is associated with better prognosis in glioma. As Yes-associated protein 1 (YAP1) is an important regulator of tumor progression, its role in glioma expressing IDH1 R132H mutation was investigated. Diminished nuclear YAP1 in IDH1 mutant patient gliomas and cell lines was accompanied by decreased TFAM levels. Luciferase reporter assays and chromatin immunoprecipitation indicated the functionality of TEAD2 site on TFAM promoter in mediating its YAP1-dependent expression. YAP1-dependent mitochondrial fragmentation and ROS generation was accompanied by decreased TERT levels and increased mitochondrial TERT localization in IDH1 R132H cells. Treatment with Bosutinib that prevents extranuclear TERT shuttle, further elevated ROS in IDH1 R132H cells and triggered apoptosis. Importantly, Bosutinib elevated ROS levels and induced apoptosis in IDH1 WT cells upon concurrent depletion of YAP1. These findings highlight the involvement of YAP1 in coupling mitochondrial dysfunction with TERT mitochondrial shuttle to constitute an essential non-canonical function of YAP1 in regulating redox homeostasis.
    Keywords:  Glioma; IDH1; Mitochondria; TERT; TFAM; YAP1
  31. Am J Transl Res. 2021 ;13(9): 9950-9973
      Cancer cells usually show adaptations to their metabolism that facilitate their growth, invasiveness, and metastasis. Therefore, reprogramming the energy metabolism is one of the current key foci of cancer research and treatment. Although aerobic glycolysis-the Warburg effect-has been thought to be the dominant energy metabolism in cancer, recent data indicate a different possibility, specifically that oxidative phosphorylation (OXPHOS) is the more likely form of energy metabolism in some cancer cells. Due to the heterogeneity of epithelial ovarian cancer, there are different metabolic preferences among cell types, study types (in vivo/in vitro), and invasiveness. Current knowledge acknowledges glycolysis to be the main energy provider in ovarian cancer growth, invasion, migration, and viability, so specific agents targeting the glycolysis or OXPHOS pathways have been used in previous studies to attenuate tumor progression and increase chemosensitization. However, chemoresistant cell lines exert various metabolic preferences. This review comprehensively summarizes the information from existing reports which could together provide an in-depth understanding and insights for the development of a novel targeted therapy which can be used as an adjunctive treatment to standard chemotherapy to decelerate tumor progression and decrease the epithelial ovarian cancer mortality rate.
    Keywords:  Chemoresistance; chemosensitivity; epithelial ovarian cancer; glycolysis; oxidative phosphorylation
  32. Biochem Biophys Res Commun. 2021 Oct 09. pii: S0006-291X(21)01399-1. [Epub ahead of print]581 31-37
      Glioblastoma is the most serious type of brain cancer with poor prognosis. Here, using the publicly available glioma database, we identified that USP30-AS1, an antisense lncRNA locating on the opposite strand of USP30 locus, is upregulated in human gliomas, particularly in high grade glioma. High level of USP30-AS1 is correlated with poor survival in both primary and recurrent glioma patients. USP30-AS1 regulates mitochondrial homeostasis and mitophagy in glioblastoma cells. Knockdown of USP30-AS1 decreases mitochondrial protein expression and mitochondrial mass, promotes mitochondrial uncoupler-induced mitophagy. However, USP30-AS1 does not regulate USP30 expression in a cis-regulatory manner. In summary, this study proposed that USP30-AS1 may serve as a valuable prognostic marker for gliomas. USP3-AS1 is a negative regulator of mitophagy and the regulatory effect is USP30-independent. USP30-AS1 mediated repression of mitophagy may contribute to the loss of mitochondrial homeostasis and tumor development in glioma.
    Keywords:  Glioblastoma; LncRNA; Mitochondria; Mitophagy; USP30-AS1
  33. J Int Med Res. 2021 Oct;49(10): 3000605211044368
      OBJECTIVES: The anti-cancer activity of doxycycline has been reported in many cancers but not renal cell carcinoma (RCC). This study aimed to determine the efficacy of doxycycline alone and in combination with paclitaxel and analyze the underlying mechanism in RCC.METHODS: Proliferation, colony formation and apoptosis assays were performed in RCC cell lines after drug treatments. An RCC xenograft mouse model was generated, and tumor growth was monitored. Mechanistic studies focused on mitochondrial translation and functions.
    RESULTS: Doxycycline at clinically achievable concentrations inhibited proliferation and colony formation and induced apoptosis in RCC cell lines. In normal kidney cells, doxycycline at the same concentrations either had no effect or was less effective. The combination index value demonstrated that doxycycline and paclitaxel were synergistic in vitro. Consistently, this combination therapy was significantly more effective than the monotherapy in RCC xenograft mice without causing significant toxicity. Mechanistic studies revealed that doxycycline acts on RCC cells via preferentially inhibiting mitochondrial DNA translation, thereby disrupting multiple mitochondrial complexes and impairing mitochondrial respiration.
    CONCLUSIONS: Doxycycline is a useful addition to the treatment strategy for RCC. Our work also highlights the therapeutic value of mitochondrial translation inhibition in sensitizing RCC to chemotherapy.
    Keywords:  Renal cell carcinoma; chemotherapy; doxycycline; mitochondrial translation; paclitaxel; synergism
  34. Nat Commun. 2021 Oct 14. 12(1): 6011
      Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role.
  35. Nature. 2021 Oct 13.
      Human epithelial tissues accumulate cancer-driver mutations with age1-9, yet tumour formation remains rare. The positive selection of these mutations suggests that they alter the behaviour and fitness of proliferating cells10-12. Thus, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less competitive neighbours11-14. However, little is known about how such dynamic competition in normal epithelia influences early tumorigenesis. Here we show that the majority of newly formed oesophageal tumours are eliminated through competition with mutant clones in the adjacent normal epithelium. We followed the fate of nascent, microscopic, pre-malignant tumours in a mouse model of oesophageal carcinogenesis and found that most were rapidly lost with no indication of tumour cell death, decreased proliferation or an anti-tumour immune response. However, deep sequencing of ten-day-old and one-year-old tumours showed evidence of selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased early tumour removal, whereas pharmacological inhibition of clonal competition reduced tumour loss. These results support a model in which survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the surrounding normal tissue. Mutant clones in normal epithelium have an unexpected anti-tumorigenic role in purging early tumours through cell competition, thereby preserving tissue integrity.
  36. Front Cell Dev Biol. 2021 ;9 719209
      ATP-binding cassette (ABC) transporter family are major contributors to the drug resistance establishment of breast cancer cells. Breast cancer resistant protein (BCRP), one of the ABC transporters, has long been recognized as a pump that effluxes the therapeutic drugs against the concentration gradient. However, recent studies suggest that the biological function of BCRP is not limited in its drug pump activity. Herein, the role of BCRP in the proliferation and survival of drug-resistant breast cancer cells was investigated. We found that BCRP is not the major drug pump to efflux epirubicin in the resistant cells that express multiple ABC transporters. Silencing of BCRP significantly impairs cell proliferation and induces apoptosis of the resistant cells in vitro and in vivo. RNA-sequencing and high-throughput proteomics suggest that BCRP is an inhibitory factor of oxidative phosphorylation (OXPHOS). Further research suggests that BCRP is localized in the mitochondria of the resistant cells. Knockdown of BCRP elevated the intracellular reactive oxygen species level and eventually promotes the cell to undergo apoptosis. This study demonstrated that BCRP exerts important onco-promoting functions in the drug-resistant breast cancer cells independent of its well-recognized drug efflux activity, which shed new light on understanding the complex functional role of ABC transporters in drug-resistant cells.
    Keywords:  breast cancer resistant protein; drug-resistant breast cancer; proliferation; reactive oxygen species; survival
  37. Cancer Discov. 2021 Oct 14.
      Metastasis is an inefficient process in which the vast majority of cancer cells are fated to die, partly because they experience oxidative stress. Metastasizing cancer cells migrate through diverse environments that differ dramatically from their tumor of origin, leading to redox imbalances. The rare metastasizing cells that survive undergo reversible metabolic changes that confer oxidative stress resistance. We review the changes in redox regulation that cancer cells undergo during metastasis. By better understanding these mechanisms, it may be possible to develop pro-oxidant therapies that block disease progression by exacerbating oxidative stress in cancer cells. SIGNIFICANCE: Oxidative stress often limits cancer cell survival during metastasis, raising the possibility of inhibiting cancer progression with pro-oxidant therapies. This is the opposite strategy of treating patients with antioxidants, an approach that worsened outcomes in large clinical trials.
  38. Cell. 2021 Oct 07. pii: S0092-8674(21)01105-3. [Epub ahead of print]
      Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover "subTMEs," histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. "Reactive" subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich "deserted" subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.
    Keywords:  cancer-associated fibroblasts; pancreatic cancer; patient-derived organoids; proteomics; stromal heterogeneity; systems biology; treatment resistance; tumor microenvironment
  39. Mol Cancer Res. 2021 Oct 11. pii: molcanres.MCR-21-0314-E.2021. [Epub ahead of print]
      FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), with the most common mutation being internal tandem duplications (ITDs). The presence of FLT3-ITD in AML carries a particularly poor prognosis and renders therapeutic resistance. New druggable targets are thus needed in this disease. In this study, we demonstrate the effects of de novo creatine biosynthesis upregulation by FLT3-ITD on AML sustainability. Our data show that FLT3-ITD constitutively activates the STAT5 signaling pathway, which upregulates the expression of glycine amidinotransferase (GATM), the first rate-limiting enzyme of de novo creatine biosynthesis. Pharmacological FLT3-ITD inhibition reduces intracellular creatinine levels through transcriptional down-regulation of genes in the de novo creatine biosynthesis pathway. The same reduction can be achieved by cyclocreatine or genetic GATM knock down with shRNA and is reflected in significant decrease of cell proliferation and moderate increase of cell apoptosis in FLT3-ITD mutant cell lines. Those effects are at least partially mediated through the AMPK/mTOR signaling pathway. This study uncovers a previously uncharacterized role of creatine metabolic pathway in the maintenance of FLT3-ITD mutant AML and suggests that targeting this pathway may serve as a promising therapeutic strategy for FLT3-ITD positive AML. Implications: FLT3-ITD mutation in AML upregulates de novo creatine biosynthesis that we show can be suppressed to diminish the proliferation and survival of blast cells.
  40. Cancers (Basel). 2021 Oct 08. pii: 5033. [Epub ahead of print]13(19):
      Obesity contributes to ovarian cancer (OC) progression via tumorigenic chemokines. Adipocytes and OC cells highly express CXCR2, and its ligands CXCL1/8, respectively, indicating that the CXCL1/8-CXCR2 axis is a molecular link between obesity and OC. Here, we investigated how the adipocyte-specific CXCR2 conditional knockout (cKO) affected the peritoneal tumor microenvironment of OC in a high-fat diet (HFD)-induced obese mouse model. We first generated adipocyte-specific CXCR2 cKO in mice: adipose tissues were not different in crown-like structures and adipocyte size between the wild-type (WT) and cKO mice but expressed lower levels of CCL2/6 compared to the obese WT mice. HFD-induced obese mice had a shorter survival time than lean mice. Particularly, obese WT and cKO mice developed higher tumors and ascites burdens, respectively. The ascites from the obese cKO mice showed increased vacuole clumps but decreased the floating tumor burden, tumor-attached macrophages, triglyceride, free fatty acid, CCL2, and TNF levels compared to obese WT mice. A tumor analysis revealed that obese cKO mice attenuated inflammatory areas, PCNA, and F4/80 compared to obese WT mice, indicating a reduced tumor burden, and there were positive relationships between the ascites and tumor parameters. Taken together, the adipocyte-specific CXCR2 cKO was associated with obesity-induced ascites despite a reduced tumor burden, likely altering the peritoneal tumor microenvironment of OC.
    Keywords:  CXCR2; ascites; high-fat diet; obesity; ovarian cancer
  41. Mol Cell. 2021 Oct 04. pii: S1097-2765(21)00749-8. [Epub ahead of print]
      Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.
    Keywords:  TMTpro tags; multiplexed quantitative proteomics; protein degradation; protein half-lives; short-lived proteins
  42. Theranostics. 2021 ;11(19): 9705-9720
      Metastasis is the major cause of high mortality in lung cancer. Exploring the underlying mechanisms of metastasis thus holds promise for identifying new therapeutic strategies that may enhance survival. Methods: We applied quantitative mass spectrometry to compare protein expression profiles between primary and metastatic lung cancer cells whilst investigating metastasis-related molecular features. Results: We discovered that BCAT1, the key enzyme in branched-chain amino acid metabolism, is overexpressed at the protein level in metastatic lung cancer cells, as well as in metastatic tissues from lung cancer patients. Analysis of transcriptomic data available in the TCGA database revealed that increased BCAT1 transcription is associated with poor overall survival of lung cancer patients. In accord with a critical role in metastasis, shRNA-mediated knockdown of BCAT1 expression reduced migration of metastatic cells in vitro and the metastasis of these cells to distal organs in nude mice. Mechanistically, high levels of BCAT1 depleted α-ketoglutarate (α-KG) and promoted expression of SOX2, a transcription factor regulating cancer cell stemness and metastasis. Conclusion: Our findings suggest that BCAT1 plays an important role in promoting lung cancer cell metastasis, and may define a novel pathway to target as an anti-metastatic therapy.
    Keywords:  BCAT1; SOX2; metastasis; stemness
  43. Biol Open. 2021 Oct 13. pii: bio.059072. [Epub ahead of print]
      Next-generation sequencing can quickly reveal genetic variation potentially linked to heritable disease. As databases encompassing human variation continue to expand, rare variants have been of high interest, since the frequency of a variant is expected to be low if the genetic change leads to a loss of fitness or fecundity. However, the use of variant frequency when seeking genomic changes linked to disease remains very challenging. Here, we explore the role of selection in controlling human variant frequency using the HelixMT database, which encompasses hundreds of thousands of mitochondrial DNA (mtDNA) samples. We find that a substantial number of synonymous substitutions, which have no effect on protein sequence, were never encountered in this large study, while many other synonymous changes are found at very low frequencies. Further analyses of human and mammalian mtDNA datasets indicate that the population frequency of synonymous variants is predominantly determined by mutational biases rather than by strong selection acting upon nucleotide choice. Our work has important implications that extend to the interpretation of variant frequency for non-synonymous substitutions.
    Keywords:  Genomic variation; Mitochondrial DNA; Mutational bias; Pathogenicity prediction; Population frequency
  44. Nat Metab. 2021 Oct 14.
      Macrophages rely on tightly integrated metabolic rewiring to clear dying neighboring cells by efferocytosis during homeostasis and disease. Here we reveal that glutaminase-1-mediated glutaminolysis is critical to promote apoptotic cell clearance by macrophages during homeostasis in mice. In addition, impaired macrophage glutaminolysis exacerbates atherosclerosis, a condition during which, efficient apoptotic cell debris clearance is critical to limit disease progression. Glutaminase-1 expression strongly correlates with atherosclerotic plaque necrosis in patients with cardiovascular diseases. High-throughput transcriptional and metabolic profiling reveals that macrophage efferocytic capacity relies on a non-canonical transaminase pathway, independent from the traditional requirement of glutamate dehydrogenase to fuel ɑ-ketoglutarate-dependent immunometabolism. This pathway is necessary to meet the unique requirements of efferocytosis for cellular detoxification and high-energy cytoskeletal rearrangements. Thus, we uncover a role for non-canonical glutamine metabolism for efficient clearance of dying cells and maintenance of tissue homeostasis during health and disease in mouse and humans.