bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2023‒03‒26
thirty-two papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Biol Chem. 2023 Mar 24.
      The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.
    Keywords:  NDUFB10; OXPHOS; complex I; mitochondria; respiratory chain supercomplexes
  2. J Biol Chem. 2023 Mar 17. pii: S0021-9258(23)00266-1. [Epub ahead of print] 104624
      Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain (ETC), which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as ETC inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment, but do not act on ROS released to the intermembrane space. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth in vivo. Stable expression of non-degradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.
    Keywords:  Hypoxia-Inducible Factor-1; Reactive oxygen species; cancer; mitochondrial intermembrane space; peroxiredoxin-5; redox signaling
  3. Genes Nutr. 2023 Mar 24. 18(1): 5
      BACKGROUND: Serine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein's active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.RESULTS: Homozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.
    CONCLUSIONS: The results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.
    Keywords:  Energy metabolism; Folate; One-carbon metabolism; Oxygen consumption rate; Pyruvate kinase; SHMT2
  4. Front Oncol. 2023 ;13 1129352
      Introduction: Ovarian cancer is one of the leading causes of death for women with cancer worldwide. Energy requirements for tumor growth in epithelial high-grade serous ovarian cancer (HGSOC) are fulfilled by a combination of aerobic glycolysis and oxidative phosphorylation (OXPHOS). Although reduced OXPHOS activity has emerged as one of the significant contributors to tumor aggressiveness and chemoresistance, up-regulation of mitochondrial antioxidant capacity is required for matrix detachment and colonization into the peritoneal cavity to form malignant ascites in HGSOC patients. However, limited information is available about the mitochondrial biogenesis regulating OXPHOS capacity and generation of mitochondrial reactive oxygen species (mtROS) in HGSOC.Methods: To evaluate the modulation of OXPHOS in HGSOC tumor samples and ovarian cancer cell lines, we performed proteomic analyses of proteins involved in mitochondrial energy metabolism and biogenesis and formation of mtROS by immunoblotting and flow cytometry, respectively.
    Results and discussion: We determined that the increased steady-state expression levels of mitochondrial- and nuclear-encoded OXPHOS subunits were associated with increased mitochondrial biogenesis in HGSOC tumors and ovarian cancer cell lines. The more prominent increase in MT-COII expression was in agreement with significant increase in mitochondrial translation factors, TUFM and DARS2. On the other hand, the ovarian cancer cell lines with reduced OXPHOS subunit expression and mitochondrial translation generated the highest levels of mtROS and significantly reduced SOD2 expression. Evaluation of mitochondrial biogenesis suggested that therapies directed against mitochondrial targets, such as those involved in transcription and translation machineries, should be considered in addition to the conventional chemotherapies in HGSOC treatment.
    Keywords:  MT-COII; TFAM; TUFM; high-grade serous ovarian cancer (HGSOC); mitochondrial biogenesis; mitochondrial reactive oxygen species (mtROS); mitochondrial translation and transcription; oxidative phosphorylation (OXPHOS)
  5. Elife. 2023 Mar 23. pii: e84415. [Epub ahead of print]12
      Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.
    Keywords:  complex I; drosophila melanogaster; electron transport chain; mitochondria; molecular biophysics; respiration; single particle cryoEM; structural biology
  6. PLoS Comput Biol. 2023 Mar 23. 19(3): e1010953
      Mitochondria are highly dynamic organelles, containing vital populations of mitochondrial DNA (mtDNA) distributed throughout the cell. Mitochondria form diverse physical structures in different cells, from cell-wide reticulated networks to fragmented individual organelles. These physical structures are known to influence the genetic makeup of mtDNA populations between cell divisions, but their influence on the inheritance of mtDNA at divisions remains less understood. Here, we use statistical and computational models of mtDNA content inside and outside the reticulated network to quantify how mitochondrial network structure can control the variances of inherited mtDNA copy number and mutant load. We assess the use of moment-based approximations to describe heteroplasmy variance and identify several cases where such an approach has shortcomings. We show that biased inclusion of one mtDNA type in the network can substantially increase heteroplasmy variance (acting as a genetic bottleneck), and controlled distribution of network mass and mtDNA through the cell can conversely reduce heteroplasmy variance below a binomial inheritance picture. Network structure also allows the generation of heteroplasmy variance while controlling copy number inheritance to sub-binomial levels, reconciling several observations from the experimental literature. Overall, different network structures and mtDNA arrangements within them can control the variances of key variables to suit a palette of different inheritance priorities.
  7. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531918. [Epub ahead of print]
      Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anti-cancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets-cysteine C53 of HIG2DA and lysine K72 of COX5A-that are part of complex IV of the electron transport chain and contributed significantly to the anti-proliferative activity. OPA activated mitochondrial respiration in a HIG2DA and COX5A-dependent manner, led to an initial spike in mitochondrial ATP, but then compromised mitochondrial membrane potential leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anti-cancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.
  8. Nature. 2023 Mar 22.
      Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
  9. J Cell Sci. 2023 Mar 21. pii: jcs.260049. [Epub ahead of print]
      Glucose sensing in pancreatic beta-cells depends on oxidative phosphorylation and mitochondria-derived signals that promote insulin secretion. Using mass spectrometry-based phosphoproteomics to search for down-stream effectors of glucose dependent signal transduction in INS-1E insulinoma cells, we identified the outer mitochondrial membrane protein SLC25A46. Under resting glucose concentrations, SLC25A46 was phosphorylated on a pair of threonine residues (T44/T45) and was dephosphorylated in response to glucose-induced calcium signals. Overexpression of SLC25A46 in INS-1E cells caused complete mitochondrial fragmentation, resulting in a mild mitochondrial defect associated with lowered glucose-induced insulin secretion. In contrast, inactivation of the SLC25A46 gene resulted in dramatic mitochondrial hyperfusion but without affecting respiratory activity or insulin secretion. Consequently, SLC25A46 is not essential for metabolism-secretion coupling under normal nutrient conditions. Importantly, insulin secreting cells lacking SLC25A46 had an exacerbated sensitivity to lipotoxic conditions undergoing massive apoptosis when exposed to palmitate. Therefore, in addition to its role in mitochondrial dynamics, SLC25A46 plays a role in preventing mitochondria-induced apoptosis in INS-E cells exposed to nutrient stress. By protecting mitochondria, SLC25A46 may help to maintain beta-cell mass essential for blood glucose control.
    Keywords:  Beta-cell; Mitochondria; Mitochondrial dynamics
  10. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]
      Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.
  11. bioRxiv. 2023 Mar 08. pii: 2023.03.06.531392. [Epub ahead of print]
      Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here, we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mt-genome in different tissues throughout aging. We used ultra-sensitive Duplex Sequencing to profile ∼2.5 million mt-genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloging ∼1.2 million mitochondrial somatic mutations. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the Light Strand Origin of Replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared to primates with a surfeit of reactive oxygen species-associated G>T/C>A mutations and that somatic mutations in protein coding genes exhibit strong signatures of positive selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that "re-align" mito-nuclear ancestry within an organism's lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.
  12. Front Physiol. 2023 ;14 1114595
      Methods of isolating mitochondria commonly utilise mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. Here, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation. This protocol results in high-yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate the enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from murine skeletal muscle cell lines (C2C12) and tibialis anterior tissue have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. Our method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.
    Keywords:  cavitation; methodolody; mitochondria; protocol; skeletal muscle
  13. Biomed Pharmacother. 2023 Mar 20. pii: S0753-3322(23)00312-8. [Epub ahead of print]161 114524
      Prostate and ovarian cancers affect the male and female reproductive organs and are among the most common cancers in developing countries. Previous studies have demonstrated that cancer cells have a high rate of aerobic glycolysis that is present in nearly all invasive human cancers and persists even under normoxic conditions. Aerobic glycolysis has been correlated with chemotherapeutic resistance and tumor aggressiveness. These data suggest that mitochondrial dysfunction may confer a significant proliferative advantage during the somatic evolution of cancer. In this study we investigated the effect of direct mitochondria transplantation on cancer cell proliferation and chemotherapeutic sensitivity in prostate and ovarian cancer models, both in vitro and in vivo. Our results show that the transplantation of viable, respiration competent mitochondria has no effect on cancer cell proliferation but significantly decreases migration and alters cell cycle checkpoints. Our results further demonstrate that mitochondrial transplantation significantly increases chemotherapeutic sensitivity, providing similar apoptotic levels with low-dose chemotherapy as that achieved with high-dose chemotherapy. These results suggest that mitochondria transplantation provides a novel approach for early prostate and ovarian cancer therapy, significantly increasing chemotherapeutic sensitivity in in vitro and in vivo murine models.
    Keywords:  Cisplatin; Docetaxel; Mitochondrial transplantation; Ovarian cancer; Paclitaxel; Prostate cancer
  14. Proc Natl Acad Sci U S A. 2023 Mar 28. 120(13): e2210796120
      Rewiring of redox metabolism has a profound impact on tumor development, but how the cellular heterogeneity of redox balance affects leukemogenesis remains unknown. To precisely characterize the dynamic change in redox metabolism in vivo, we developed a bright genetically encoded biosensor for H2O2 (named HyPerion) and tracked the redox state of leukemic cells in situ in a transgenic sensor mouse. A H2O2-low (HyPerion-low) subset of acute myeloid leukemia (AML) cells was enriched with leukemia-initiating cells, which were endowed with high colony-forming ability, potent drug resistance, endosteal rather than vascular localization, and short survival. Significantly high expression of malic enzymes, including ME1/3, accounted for nicotinamide adenine dinucleotide phosphate (NADPH) production and the subsequent low abundance of H2O2. Deletion of malic enzymes decreased the population size of leukemia-initiating cells and impaired their leukemogenic capacity and drug resistance. In summary, by establishing an in vivo redox monitoring tool at single-cell resolution, this work reveals a critical role of redox metabolism in leukemogenesis and a potential therapeutic target.
    Keywords:  AML; H2O2 biosensor; drug resistance; leukemia-initiating cells; redox metabolism
  15. J Transl Med. 2023 Mar 25. 21(1): 218
      BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear.METHODS: HepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives.
    RESULTS: Here,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. N-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress.
    CONCLUSION: These data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.
    Keywords:  HCC; Mitochondria; Mitophagy; NOX4; Nrf2; ROS
  16. Technol Cancer Res Treat. 2023 Jan-Dec;22:22 15330338231161141
      Introduction: Nuclear respiratory factor 1 (NRF1) is an important regulator involved in mitochondrial biogenesis and energy metabolism. However, the specific mechanism of NRF1 in anoikis and epithelial-mesenchymal transition (EMT) remains unclear. Methods: We examined the effect of NRF1 on mitochondria and identified the specific mechanism through transcriptome sequencing, and explored the relationships among NRF1, anoikis, and EMT. Results: We found that upregulated NRF1 expression led to increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP generation. Simultaneously, a significant amount of ROS is generated during OXPHOS. Alternatively, NRF1 upregulates the expression of ROS-scavenging enzymes, allowing tumor cells to maintain low ROS levels and promoting anoikis resistance and EMT. We also found that exogenous ROS was maintained at a low level by NRF1 in breast cancer cells. Conclusion: our study provides mechanistic insight into the function of NRF1 in breast cancer, indicating that NRF1 may serve as a therapeutic target for breast cancer treatment.
    Keywords:  EMT; NRF1; OXPHOS; ROS; anoikis; breast cancer
  17. Cell Rep. 2023 Mar 22. pii: S2211-1247(23)00314-5. [Epub ahead of print]42(4): 112303
      Oncogenes destabilize STING in epithelial cell-derived cancer cells, such as head and neck squamous cell carcinomas (HNSCCs), to promote immune escape. Despite the abundance of tumor-infiltrating myeloid cells, HNSCC presents notable resistance to STING stimulation. Here, we show how saturated fatty acids in the microenvironment dampen tumor response to STING stimulation. Using single-cell analysis, we found that obesity creates an IFN-I-deprived tumor microenvironment with a massive expansion of suppressive myeloid cell clusters and contraction of effector T cells. Saturated fatty acids, but not unsaturated fatty acids, potently inhibit the STING-IFN-I pathway in HNSCC cells. Myeloid cells from obese mice show dampened responses to STING stimulation and are more suppressive of T cell activation. In agreement, obese hosts exhibited increased tumor burden and lower responsiveness to STING agonist. As a mechanism, saturated fatty acids induce the expression of NLRC3, depletion of which results in a T cell inflamed tumor microenvironment and IFN-I-dependent tumor control.
    Keywords:  CP: Cancer; CP: Immunology; NLRC3; STING; head and neck cancer; immunogenicity; innate immunity; metabolism; obesity; saturated fatty acids; type-I interferon
  18. bioRxiv. 2023 Mar 11. pii: 2023.03.11.532189. [Epub ahead of print]
      Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, it's unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H 2 O 2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H 2 O 2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.
  19. J Nutr Biochem. 2023 Mar 22. pii: S0955-2863(23)00055-4. [Epub ahead of print] 109322
      Vitamin D receptor (VDR) is an essential transcription factor (TF) synthesized in different cell types. We hypothesized that VDR might also act as a mitochondrial TF. We conducted the experiments in primary cortical neurons, PC12, HEK293T, SH-SY5Y cell lines, human peripheral blood mononuclear cells (PBMC) and human brain. We showed that vitamin D/VDR affects the expression of mitochondrial DNA (mtDNA) encoded oxidative phosphorylation (OXPHOS) subunits. We observed the co-localization of VDR with mitochondria and the mtDNA with confocal microscopy. mtDNA-chromatin-immunoprecipitation and electrophoretic mobility shift assays indicated that VDR was able to bind to the mtDNA D-loop site in several locations, with a consensus sequence 'MMHKCA'. We also reported the possible interaction between VDR and mitochondrial transcription factor A (TFAM) and their binding sites located in close proximity in mtDNA. Consequently, our results showed for the first time that VDR was able to bind and regulate mtDNA transcription and interact with TFAM even in the human brain. These results not only revealed a novel function of VDR, but also showed that VDR is indispensable for energy demanded cells.
    Keywords:  Vitamin D; mitochondria; mitochondrial DNA; mitochondrial transcription factor; transcription factor; vitamin D receptor
  20. FASEB J. 2023 Apr;37(4): e22853
      Obesity is characterized by systemic low-grade inflammation associated with disturbances of intestinal homeostasis and microbiota dysbiosis. Mitochondrial metabolism sustains epithelial homeostasis by providing energy to colonic epithelial cells (CEC) but can be altered by dietary modulations of the luminal environment. Our study aimed at evaluating whether the consumption of an obesogenic diet alters the mitochondrial function of CEC in mice. Mice were fed for 22 weeks with a 58% kcal fat diet (diet-induced obesity [DIO] group) or a 10% kcal fat diet (control diet, CTRL). Colonic crypts were isolated to assess mitochondrial function while colonic content was collected to characterize microbiota and metabolites. DIO mice developed obesity, intestinal hyperpermeability, and increased endotoxemia. Analysis of isolated colonic crypt bioenergetics revealed a mitochondrial dysfunction marked by decreased basal and maximal respirations and lower respiration linked to ATP production in DIO mice. Yet, CEC gene expression of mitochondrial respiration chain complexes and mitochondrial dynamics were not altered in DIO mice. In parallel, DIO mice displayed increased colonic bile acid concentrations, associated with higher abundance of Desulfovibrionaceae. Sulfide concentration was markedly increased in the colon content of DIO mice. Hence, chronic treatment of CTRL mouse colon organoids with sodium sulfide provoked mitochondrial dysfunction similar to that observed in vivo in DIO mice while acute exposure of isolated mitochondria from CEC of CTRL mice to sodium sulfide diminished complex IV activity. Our study provides new insights into colon mitochondrial dysfunction in obesity by revealing that increased sulfide production by DIO-induced dysbiosis impairs complex IV activity in mouse CEC.
    Keywords:  high fat diet; intestine; microbiota; mitochondria; obesity
  21. Commun Biol. 2023 Mar 22. 6(1): 307
      In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.
  22. J Thorac Oncol. 2023 Mar 21. pii: S1556-0864(23)00197-1. [Epub ahead of print]
      INTRODUCTION: In KRAS-mutant non-small lung cancer (NSCLC), co-occurring alterations in LKB1 confer a negative prognosis compared to other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetical inhibition of HDAC6 demonstrated impaired activity of numerous enzymes involved in glycolysis. Based on these prior findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors.EXPERIMENTAL DESIGN: Using cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 (KP) mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo.
    RESULTS: Surprisingly, KL-mutant cells demonstrated reduced levels of redox-sensitive cofactors at baseline. This associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and anti-tumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo.
    CONCLUSIONS: Exploring the differential metabolism of KL and KP mutant NSCLC, we identified decreased metabolic reserve in KL mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC based on a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.
    Keywords:  HDAC6; KRAS; LKB1; TP53; glutaminase inhibition; glycolysis; non-small cell lung cancer; oxidative stress
  23. Sci Adv. 2023 Mar 24. 9(12): eadd9554
      Isoenzyme divergence is a prevalent mechanism governing tissue-specific and developmental stage-specific metabolism in mammals. The lactate dehydrogenase (LDH) isoenzyme spectrum reflects the tissue-specific metabolic status. We found that three tetrameric isoenzymes composed of LDHA and LDHB (LDH-3/4/5) comprise the LDH spectrum in T cells. Genetically deleting LDHA or LDHB altered the isoenzyme spectrum by removing all heterotetramers and leaving T cells with LDH-1 (the homotetramer of LDHB) or LDH-5 (the homotetramer of LDHA), respectively. Accordingly, deleting LDHA suppressed glycolysis, cell proliferation, and differentiation. Unexpectedly, deleting LDHB enhanced glycolysis but suppressed T cell differentiation, indicating that an optimal zone of glycolytic activity is required to maintain cell fitness. Mechanistically, the LDH isoenzyme spectrum imposed by LDHA and LDHB is necessary to optimize glycolysis to maintain a balanced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide hydrogen pool. Our results suggest that the LDH isoenzyme spectrum enables "Goldilocks levels" of glycolytic and redox activity to control T cell differentiation.
  24. Trends Immunol. 2023 Mar 16. pii: S1471-4906(23)00045-5. [Epub ahead of print]
      Mitochondrial outer membrane permeabilization (MOMP) is crucial for the cytosolic accumulation of mitochondrial DNA (mtDNA) species that are required to jumpstart innate and adaptive immunity. Recent data reported by Ghosh et al. suggest that tumor protein p53 regulates MOMP-dependent type I interferon (IFN) production, not only via MOMP-promoting effects, but also by directing mtDNA-degrading exonucleases to proteasomal processing.
    Keywords:  BAX; BCL2; DNA-damage response; TRIM24; apoptosis
  25. bioRxiv. 2023 Mar 12. pii: 2023.03.11.532186. [Epub ahead of print]
      The integrated stress response (ISR) is a network of eIF2 α kinases, comprising PERK, GCN2, HRI, and PKR, that induce translational and transcriptional signaling in response to diverse insults. The PERK ISR kinase regulates mitochondria in response to endoplasmic reticulum (ER) stress. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activators of other ISR kinases to rescue ISR signaling and promote mitochondrial adaptation in cells lacking PERK. We show that the HRI activator BtdCPU and the GCN2 activator halofuginone activate ISR signaling and restore ER stress sensitivity in Perk- deficient cells. However, these compounds differentially impact mitochondria. BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and ISR activation through the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and altered mitochondrial respiration, mimicking the regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK activity and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.
  26. bioRxiv. 2023 Mar 10. pii: 2023.03.08.531740. [Epub ahead of print]
      Epithelial Ovarian Cancer (EOC) is the most lethal gynecologic cancer with limited genetic alterations identified that can be therapeutically targeted. In tumor bearing mice, short-term fasting, fasting mimicking diet and calorie restriction enhance the activity of antineoplastic treatment by modulating systemic metabolism and boosting anti-tumor immunity. We tested the outcome of sixteen-hour intermittent fasting (IF) on mouse EOC progression with focus on fasting driven antitumor immune responses. IF resulted in consistent decrease of tumor promoting metabolic growth factors and cytokines, recapitulating changes that creates a tumor antagonizing environment. Immune profiling revealed that IF profoundly reshapes anti-cancer immunity by inducing increase in CD4 + and CD8 + cells, paralleled by enhanced antitumor Th1 and cytotoxic responses, by enhancing their metabolic fitness. Metabolic studies revealed that IF generated bioactive metabolite BHB which can be a potential substitute for simulating the antitumor benefits of IF. However, in a direct comparison, IF surpassed exogenous BHB therapy in improving survival and activating anti-tumor immune response. Thus, our data provides strong evidence for IF and its metabolic mediator BHB for ameliorating EOC progression and as a viable approach in maintaining and sustaining an effective anti-tumor T cell response.
  27. Elife. 2023 Mar 23. pii: e85289. [Epub ahead of print]12
      Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacologic neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacologic suppression.
    Keywords:  cell biology; mouse
  28. Aging Cell. 2023 Mar 20. e13822
      Mitochondrial dysfunction is considered a highly conserved hallmark of ageing. However, most of the studies in both model and non-model organisms are cross-sectional in design; therefore, little is known, at the individual level, on how mitochondrial function changes with age, its link to early developmental conditions or its relationship with survival. Here we manipulated the postnatal growth in zebra finches (Taeniopygia guttata) via dietary modification that induced accelerated growth without changing adult body size. In the same individuals, we examined blood cells mitochondrial functioning (mainly erythrocytes) when they were young (ca. 36 weeks) and again in mid-aged (ca. 91 weeks) adulthood. Mitochondrial function was strongly influenced by age but not by postnatal growth conditions. Across all groups, within individual ROUTINE respiration, OXPHOS and OXPHOS coupling efficiency significantly declined with age, while LEAK respiration increased. However, we found no link between mitochondrial function and the probability of survival into relatively old age (ca. 4 years). Our results suggest that the association between accelerated growth and reduced longevity, evident in this as in other species, is not attributable to age-related changes in any of the measured mitochondrial function traits.
    Keywords:  ageing; avian models; mitochondria; red blood cells; senescence; survival
  29. Trends Cancer. 2023 Mar 21. pii: S2405-8033(23)00030-4. [Epub ahead of print]
      New findings by Altea-Manzano et al. demonstrate that primary breast tumours or a high-fat diet induce palmitate secretion by alveolar type 2 (AT2) cells to prepare a premetastatic niche that promotes lung metastasis. Palmitate is utilised by cancer cells via carnitine palmitoyltransferase 1a (CPT1a) and lysine acetyltransferase 2a (KAT2a) to acetylate nuclear factor-κB (NF-κB)/p65. Blocking these enzymes reduced metastasis formation in both lean and high-fat diet mice.
    Keywords:  NF-kB; breast cancer; lung metastasis; palmitate; premetastatic niche; protein acetylation
  30. Front Immunol. 2023 ;14 1093974
      Background: Succinate dehydrogenase (SDH), one of the key enzymes in the tricarboxylic acid cycle, is mainly found in the mitochondria. SDH consists of four subunits encoding SDHA, SDHB, SDHC, and SDHD. The biological function of SDH is significantly related to cancer progression. Colorectal cancer (CRC) is one of the most common malignant tumors globally, whose most common histological subtype is colon adenocarcinoma (COAD). However, the correlation between SDH factors and COAD remains unclear.Methods: The data on pan-cancer was obtained from The Cancer Genome Atlas (TCGA) database. Kaplan-Meier survival analysis showed the prognostic ability of SDHs. The cBioPortal database reflected genetic variations of SDHs. The correlation analysis was conducted between SDHs and mitochondrial energy metabolism genes (MMGs) and the protein-protein interaction (PPI) network was built. Consequently, Univariate and Multivariate Cox Regression Analysis on SDHs and other clinical characteristics were conducted. A nomogram was established. The ssGSEA analysis visualized the association between SDHs and immune infiltration. Immunophenoscore (IPS) explored the correlation between SDHs and immunotherapy, and the correlation between SDHs and targeted therapy was investigated through Genomics of Drug Sensitivity in Cancer. Finally, qPCR and immunohistochemistry detected SDHs' expression.
    Results: After assessing SDHs differential expression in pan-cancer, we found that SDHB, SDHC, and SDHD benefit COAD patients. The cBioPortal database demonstrated that SDHA was the top gene in mutation frequency rank. Correlation analysis mirrored a strong link between SDHs and MMGs. We formulated a nomogram and found that SDHB, SDHC, SDHD, and clinical characteristics correlated with COAD patients' survival. For T helper cells, Th2 cells, and Tem, SDHA, SDHB, SDHC, and SDHD were significantly enriched in the high expression group. Moreover, COAD patients with high SDHA expression were more suitable for immunotherapy. And COAD patients with different SDHs' expression have different sensitivity to targeted drugs. Further verifying the gene and protein expression levels of SDHs, we found that the tissues were consistent with the bioinformatics analysis.
    Conclusions: Our study analyzed the expression and prognostic value of SDHs in COAD, explored the pathway mechanisms involved, and the immune cell correlations, indicating that SDHs might be biomarkers for COAD patients.
    Keywords:  colon adenocarcinoma (COAD); colorectal cancer; immune infiltration; immune treatment; prognostic; succinate dehydrogenase (SDH)
  31. Nat Commun. 2023 Mar 23. 14(1): 1615
      Single-cell RNA sequencing can reveal valuable insights into cellular heterogeneity within tumour microenvironments (TMEs), paving the way for a deep understanding of cellular mechanisms contributing to cancer. However, high heterogeneity among the same cancer types and low transcriptomic variation in immune cell subsets present challenges for accurate, high-resolution confirmation of cells' identities. Here we present scATOMIC; a modular annotation tool for malignant and non-malignant cells. We trained scATOMIC on >300,000 cancer, immune, and stromal cells defining a pan-cancer reference across 19 common cancers and employ a hierarchical approach, outperforming current classification methods. We extensively confirm scATOMIC's accuracy on 225 tumour biopsies encompassing >350,000 cancer and a variety of TME cells. Lastly, we demonstrate scATOMIC's practical significance to accurately subset breast cancers into clinically relevant subtypes and predict tumours' primary origin across metastatic cancers. Our approach represents a broadly applicable strategy to analyse multicellular cancer TMEs.
  32. Redox Biol. 2023 Mar 15. pii: S2213-2317(23)00079-4. [Epub ahead of print]62 102678
      Elevated lipid peroxidation (LPO), usually present in the tumour microenvironment (TME), is profoundly implicated in antitumour immunity and may be targeted for the development of new antitumour therapies. However, tumour cells may also rewire their metabolism to survive elevated LPO. Here, we report a novel and nonantioxidant mechanism by which tumour cells benefit from accumulated cholesterol to restrain LPO and ferroptosis, a nonapoptotic form of cell death characterized by accumulated LPO. Modulating cholesterol metabolism, especially LDLR-mediated cholesterol uptake, shifted the susceptibility of tumour cells to ferroptosis. Elevation of cellular cholesterol content specifically restrained LPO triggered by GSH-GPX4 inhibition or oxidizing factors in the TME. Furthermore, depletion of TME cholesterol by MβCD efficiently enhanced the antitumour efficacy of ferroptosis in a mouse xenograft model. Distinct from the antioxidant effect of its metabolic intermediates, the protective role of cholesterol was ascribed to its ability to decrease membrane fluidity and promote lipid raft formation, which affects the diffusion of LPO substrates. A correlation between LPO and lipid rafts was also found in tumour tissues from renal cancer patients. Together, our findings have identified a general and nonsacrificial mechanism by which cholesterol suppresses LPO, which can be exploited to enhance the efficacy of ferroptosis-based antitumour strategies.
    Keywords:  Cholesterol; Ferroptosis; LDLR; Lipid peroxidation; Lipid raft; Tumour microenvironment