bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒01‒23
thirty-six papers selected by
Kelsey Fisher-Wellman, East Carolina University



  1. Biochim Biophys Acta Bioenerg. 2022 Jan 18. pii: S0005-2728(22)00001-9. [Epub ahead of print] 148532
      The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain (RC) complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the 'respirasome free' complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.
    Keywords:  Bioenergetics; MDH2; Malate aspartate shuttle; Mitochondria; NADH redox homeostasis; Oxaloacetate; Respirasomes; Respiratory chain supercomplexes
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148532
  2. Methods Mol Biol. 2022 ;2413 55-62
      Mitochondrial metabolism plays key roles in pathologies such as cancer. The five complexes of the oxidative phosphorylation (OXPHOS) system are crucial for producing ATP and maintaining cellular functions and are particularly exploited in cancer cells. Understanding the oligomeric state of these OXPHOS complexes will help elucidate their function (or dysfunction) in cancer cells and can be used as a mechanistic tool for anticancer agents that target mitochondria. Here we describe a protocol to observe the oligomeric state of the five OXPHOS complexes by isolating mitochondrial-enriched fractions followed by assessing their oligomeric state by nondenaturing blue native page electrophoresis.
    Keywords:  Mitochondria; Native page; OXPHOS complexes; Oxidative phosphorylation
    DOI:  https://doi.org/10.1007/978-1-0716-1896-7_7
  3. J Biol Chem. 2022 Jan 18. pii: S0021-9258(22)00042-4. [Epub ahead of print] 101602
      Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation and ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. Recent high-resolution cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, and channel residues are likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the cryo-EM structure of mouse complex I with an extremely tight-binding natural-product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure-activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties ideally spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound, but stabilises the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.
    Keywords:  acetogenin; binding site; complex I; cryo-electron microscopy; inhibitor-bound structure
    DOI:  https://doi.org/10.1016/j.jbc.2022.101602
  4. Front Mol Biosci. 2021 ;8 798353
      Complex I (CI) is the largest protein complex in the mitochondrial oxidative phosphorylation electron transport chain of the inner mitochondrial membrane and plays a key role in the transport of electrons from reduced substrates to molecular oxygen. CI is composed of 14 core subunits that are conserved across species and an increasing number of accessory subunits from bacteria to mammals. The fact that adding accessory subunits incurs costs of protein production and import suggests that these subunits play important physiological roles. Accordingly, knockout studies have demonstrated that accessory subunits are essential for CI assembly and function. Furthermore, clinical studies have shown that amino acid substitutions in accessory subunits lead to several debilitating and fatal CI deficiencies. Nevertheless, the specific roles of CI's accessory subunits have remained mysterious. In this review, we explore the possible roles of each of mammalian CI's 31 accessory subunits by integrating recent high-resolution CI structures with knockout, assembly, and clinical studies. Thus, we develop a framework of experimentally testable hypotheses for the function of the accessory subunits. We believe that this framework will provide inroads towards the complete understanding of mitochondrial CI physiology and help to develop strategies for the treatment of CI deficiencies.
    Keywords:  accessory subunits; electron transport chain; mitochondrial complex I; mitochondrial diseases; oxidative phosphorylation (OXPHOS)
    DOI:  https://doi.org/10.3389/fmolb.2021.798353
  5. Sci Adv. 2022 Jan 21. 8(3): eabg6383
      Access to electron acceptors supports oxidized biomass synthesis and can be limiting for cancer cell proliferation, but how cancer cells overcome this limitation in tumors is incompletely understood. Nontransformed cells in tumors can help cancer cells overcome metabolic limitations, particularly in pancreatic cancer, where pancreatic stellate cells (PSCs) promote cancer cell proliferation and tumor growth. However, whether PSCs affect the redox state of cancer cells is not known. By taking advantage of the endogenous fluorescence properties of reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide cofactors we use optical imaging to assess the redox state of pancreatic cancer cells and PSCs and find that direct interactions between PSCs and cancer cells promote a more oxidized state in cancer cells. This suggests that metabolic interaction between cancer cells and PSCs is a mechanism to overcome the redox limitations of cell proliferation in pancreatic cancer.
    DOI:  https://doi.org/10.1126/sciadv.abg6383
  6. Oncogenesis. 2022 Jan 21. 11(1): 6
      Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41389-022-00380-z
  7. Nat Metab. 2022 Jan 20.
      Homeostasis maintains serum metabolites within physiological ranges. For glucose, this requires insulin, which suppresses glucose production while accelerating its consumption. For other circulating metabolites, a comparable master regulator has yet to be discovered. Here we show that, in mice, many circulating metabolites are cleared via the tricarboxylic acid cycle (TCA) cycle in linear proportionality to their circulating concentration. Abundant circulating metabolites (essential amino acids, serine, alanine, citrate, 3-hydroxybutyrate) were administered intravenously in perturbative amounts and their fluxes were measured using isotope labelling. The increased circulating concentrations induced by the perturbative infusions hardly altered production fluxes while linearly enhancing consumption fluxes and TCA contributions. The same mass action relationship between concentration and consumption flux largely held across feeding, fasting and high- and low-protein diets, with amino acid homeostasis during fasting further supported by enhanced endogenous protein catabolism. Thus, despite the copious regulatory machinery in mammals, circulating metabolite homeostasis is achieved substantially through mass action-driven oxidation.
    DOI:  https://doi.org/10.1038/s42255-021-00517-1
  8. Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Jan 17. pii: S1388-1981(21)00222-5. [Epub ahead of print] 159094
      Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-β-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD+ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD+ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD+ precursor supplementation in the management of BTHS patients.
    Keywords:  Barth syndrome; Cardiolipin deficiency; Mitochondrial function; NAD(+) precursors; NAD(+) redox; Nicotinamide mononucleotide
    DOI:  https://doi.org/10.1016/j.bbalip.2021.159094
  9. Redox Biol. 2022 Jan 17. pii: S2213-2317(22)00012-X. [Epub ahead of print]50 102240
      A complex interplay between the extracellular space, cytoplasm and individual organelles modulates Ca2+ signaling to impact all aspects of cell fate and function. In recent years, the molecular machinery linking endoplasmic reticulum stores to plasma membrane Ca2+ entry has been defined. However, the mechanism and pathophysiological relevance of store-independent modes of Ca2+ entry remain poorly understood. Here, we describe how the secretory pathway Ca2+-ATPase SPCA2 promotes cell cycle progression and survival by activating store-independent Ca2+ entry through plasma membrane Orai1 channels in mammary epithelial cells. Silencing SPCA2 expression or briefly removing extracellular Ca2+ increased mitochondrial ROS production, DNA damage and activation of the ATM/ATR-p53 axis leading to G0/G1 phase cell cycle arrest and apoptosis. Consistent with these findings, SPCA2 knockdown confers redox stress and chemosensitivity to DNA damaging agents. Unexpectedly, SPCA2-mediated Ca2+ entry into mitochondria is required for optimal cellular respiration and the generation of mitochondrial membrane potential. In hormone receptor positive (ER+/PR+) breast cancer subtypes, SPCA2 levels are high and correlate with poor survival prognosis. We suggest that elevated SPCA2 expression could drive pro-survival and chemotherapy resistance in cancer cells, and drugs that target store-independent Ca2+ entry pathways may have therapeutic potential in treating cancer.
    Keywords:  Ca(2+) signaling; DNA damage Response; Doxorubicin; ER+ breast cancer; Mitochondria; Oxygen consumption rate; ROS; p53
    DOI:  https://doi.org/10.1016/j.redox.2022.102240
  10. Life (Basel). 2021 Dec 28. pii: 42. [Epub ahead of print]12(1):
      Metastasis arises owing to tumor cells' capacity to evade pro-apoptotic signals. Anoikis-the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells' ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.
    Keywords:  AMPK; anoikis; metastasis; oxidative phosphorylation; proteasome
    DOI:  https://doi.org/10.3390/life12010042
  11. Cell Rep. 2022 Jan 18. pii: S2211-1247(21)01793-9. [Epub ahead of print]38(3): 110278
      A major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.
    Keywords:  PHGDH; PSAT1; auxotrophy; breast cancer; diet; luminal tumors; serine; tumor metabolism
    DOI:  https://doi.org/10.1016/j.celrep.2021.110278
  12. Viruses. 2022 Jan 07. pii: 103. [Epub ahead of print]14(1):
      Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate's numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.
    Keywords:  aerobic glycolysis; glioblastoma (GBM); human cytomegalovirus (HCMV); lactate; metabolism; oxidative phosphorylation (OXPHOS); reactive oxygen species; tumor microenvironment
    DOI:  https://doi.org/10.3390/v14010103
  13. Oncol Rep. 2022 Mar;pii: 55. [Epub ahead of print]47(3):
      Therapeutic approaches that target the metabolism of tumor cells have been a popular research topic in recent years. Previous studies have demonstrated that glycolysis inhibitors reduce the proliferation of non‑small cell lung cancer (NSCLC) cells by interfering with the aerobic glycolytic pathway. However, the mitochondrial oxidative phosphorylation (OXPHOS) pathway in tumor cells has also been implicated in lung cancer metabolism. Metformin, a known inhibitor of mitochondrial OXPHOS, has been indicated to reduce NSCLC morbidity and mortality in clinical studies. The present article reviewed the therapeutic effects of metformin against NSCLC, both as a single agent and combined with other anticancer treatments, in order to provide a theoretical basis for its clinical use in adjuvant therapy for NSCLC.
    Keywords:  anticancer; combination therapy; metabolism; metformin; non‑small cell lung cancer
    DOI:  https://doi.org/10.3892/or.2022.8266
  14. Open Biol. 2022 Jan;12(1): 210255
      Mutations in Parkin and PINK1 cause early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: (i) thioester intermediate formation on Parkin and (ii) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyses ubiquitin transfer to substrates. We found that His433 in the RING2 domain contributes to the catalysis of acyl transfer. In cells, the mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin (pUb) in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via pUb at these sites.
    Keywords:  Mfn2; PINK1; Parkin; mitochondria; ubiquitin
    DOI:  https://doi.org/10.1098/rsob.210255
  15. Br J Pharmacol. 2022 Jan 18.
      BACKGROUND AND PURPOSE: Multidrug resistance (MDR) is the main obstacle to cancer therapy. Ample evidence shows that ATP-binding cassette (ABC) transporters and high-energy state substantially relate to cancer drug resistance. Our previous work reported an engineered therapeutic protein named PAK, which selectively inhibited tumor progression by targeting mitochondria.EXPERIMENTAL APPROACH: Here, we studied the effects of PAK on reversing drug resistance in MDR phenotypic cells and xenograft mice models. The effects of PAK on the process of mitochondrial energy production, ABC transporters expression, and the drugs enrichment in cancer cells were further investigated. RNA-seq and co-immunoprecipitation were employed to analyze the mechanism of PAK on the redistribution of ABC transporters.
    KEY RESULTS: PAK promoted the enrichment of drugs in MDR cancer cells, thus enhancing the sensitivity of cancer cells to chemotherapy. Furthermore, PAK was colocalized in the mitochondria and initiated mitochondrial injury by selectively inhibiting the mitochondrial complex V. Besides, ABCB1 and ABCC1 were found to be redistributed from the plasma membrane to the cytoplasm through the disruption of lipid rafts, which was attributed to the low energy state and the decrease of cholesterol levels.
    CONCLUSIONS AND IMPLICATIONS: Our results revealed a previously unrecognized drug resistance reversal pattern and suggested mitochondria as a clinically relevant target for the treatment of MDR malignant tumors.
    DOI:  https://doi.org/10.1111/bph.15801
  16. Am J Physiol Cell Physiol. 2022 Jan 19.
      Mitochondria are essential to cell homeostasis, and alterations in mitochondrial distribution, segregation or turnover have been linked to complex pathologies such as neurodegenerative diseases or cancer. Understanding how these functions are coordinated in specific cell types is a major challenge to discover how mitochondria globally shape cell functionality. In this review, we will first describe how mitochondrial transport and dynamics are regulated throughout the cell cycle in yeast and in mammals. Second, we will explore the functional consequences of mitochondrial transport and partitioning on cell proliferation, fate acquisition, stemness, and on the way cells adapt their metabolism. Last, we will focus on how mitochondrial clearance programs represent a further layer of complexity for cell differentiation, or in the maintenance of stemness. Defining how mitochondrial transport, dynamics and clearance are mutually orchestrated in specific cell types may help our understanding of how cells can transition from a physiological to a pathological state.
    Keywords:  dynamics; fate acquisition; mitochondria; mitophagy; transport
    DOI:  https://doi.org/10.1152/ajpcell.00256.2021
  17. Cell Rep. 2022 Jan 18. pii: S2211-1247(21)01766-6. [Epub ahead of print]38(3): 110254
      Cancer heterogeneity and evolution are not fully understood. Here, we show that mitochondrial DNA of the normal liver shapes tumor progression, histology, and immune environment prior to the acquisition of oncogenic mutation. Using conplastic mice, we show that mtDNA dictates the expression of the mitochondrial unfolded protein response (UPRmt) in the normal liver. Activation of oncogenic mutations in UPRmt-positive liver increases tumor incidence and histological heterogeneity. Further, in a subset of UPRmt-positive mice, invasive liver cancers develop. RNA sequencing (RNA-seq) analysis of the normal liver reveals that, in this subset, the PAPP-A/DDR2/SNAIL axis of invasion pre-exists along with elevated collagen. Since PAPP-A promotes immune evasion, we analyzed the immune signature and found that their livers are immunosuppressed. Further, the PAPP-A signature identifies the immune exhausted subset of hepatocellular carcinoma (HCC) in humans. Our data suggest that mtDNA of normal liver shapes the entire liver cancer portrait upon acquisition of oncogenic mutations.
    Keywords:  DDR2; PAPP-A; UPRmt; collagen; conplastic mice; estrogen receptor; immune exhausted; liver cancer; mitochondrial UPR; sexual dimorphism
    DOI:  https://doi.org/10.1016/j.celrep.2021.110254
  18. Sci Adv. 2022 Jan 21. 8(3): eabh2635
      Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.
    DOI:  https://doi.org/10.1126/sciadv.abh2635
  19. FEBS Open Bio. 2022 Jan 21.
      Mitochondrial calcium homeostasis plays critical roles in cell survival and aerobic metabolism in eukaryotes. The calcium uniporter is a highly selective calcium ion channel consisting of several subunits. Mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) are core subunits of the calcium uniporter required for calcium uptake activity in the mitochondria. Recent 3D structure analysis of the MCU-EMRE complex reconstituted in nanodiscs revealed that the human MCU exists as a tetramer forming a channel pore, with EMRE bound to each MCU at a 1:1 ratio. However, the stoichiometry of MCU and EMRE in the mitochondria has not yet been investigated. We here quantitatively examined the protein levels of MCU and EMRE in the mitochondria from mouse tissues by using characterized antibodies and standard proteins. Unexpectedly, the number of EMRE molecules was lower than that of MCU; moreover, the ratios between MCU and EMRE were significantly different among tissues. Statistical calculations based on our findings suggest that a MCU tetramer binding to 4 EMREs may exist, but at low levels in the mitochondrial inner membrane. In brain mitochondria, the majority of MCU tetramers bind to 2 EMREs; in mitochondria in liver, kidney, and heart, MCU tetramers bind to 1 EMRE; and in kidney and heart, almost half of MCU tetramers bound to no EMRE. We propose here a novel stoichiometric model of the MCU-EMRE complex in mitochondria.
    Keywords:  Calcium uniporter; EMRE; Ion channel; MCU; Mitochondria; stoichiometry
    DOI:  https://doi.org/10.1002/2211-5463.13371
  20. Life (Basel). 2021 Dec 21. pii: 4. [Epub ahead of print]12(1):
      The myth of a "typical" mitochondrial genome (mtDNA) is a rock-hard belief in the field of genetics, at least for the animal kingdom [...].
    DOI:  https://doi.org/10.3390/life12010004
  21. J Biol Chem. 2022 Jan 17. pii: S0021-9258(22)00044-8. [Epub ahead of print] 101604
      Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ signaling and Ca2+-dependent functions and has been implicated in immunity, cancer, and organ development. SOCE-dependent cytosolic Ca2+ signals are affected by mitochondrial Ca2+ transport through several competing mechanisms. However, how these mechanisms interact in shaping Ca2+ dynamics and regulating Ca2+-dependent functions remains unclear. In a recent issue, Yoast and colleagues shed light on these questions by defining multiple roles of the mitochondrial Ca2+ uniporter (MCU) in regulating SOCE, Ca2+ dynamics, transcription, and lymphocyte activation.
    DOI:  https://doi.org/10.1016/j.jbc.2022.101604
  22. Oncogene. 2022 Jan 20.
      Advanced and aggressive prostate cancer (PCa) depends on glutamine for survival and proliferation. We have previously shown that inhibition of glutaminase 1, which catalyzes the rate-limiting step of glutamine catabolism, achieves significant therapeutic effect; however, therapy resistance is inevitable. Here we report that while the glutamine carbon is critical to PCa survival, a parallel pathway of glutamine nitrogen catabolism that actively contributes to pyrimidine assembly is equally important for PCa cells. Importantly, we demonstrate a reciprocal feedback mechanism between glutamine carbon and nitrogen pathways which leads to therapy resistance when one of the two pathways is inhibited. Combination treatment to inhibit both pathways simultaneously yields better clinical outcome for advanced PCa patients.
    DOI:  https://doi.org/10.1038/s41388-021-02155-z
  23. Cancers (Basel). 2022 Jan 06. pii: 269. [Epub ahead of print]14(2):
      BACKGROUND: Somatic mutations, copy-number variations, and genome instability of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play important roles in cancer development and metastasis. However, there is scarce information about pheochromocytomas and paragangliomas (PCCs/PGLs) formation.MATERIAL: To determine the potential roles of mtDNA alterations in sporadic PCCs/PGLs, we analyzed a panel of 26 nuclear susceptibility genes and the entire mtDNA sequence of seventy-seven human tumors, using next-generation sequencing, and compared the results with normal adrenal medulla tissues. We also performed an analysis of copy-number alterations, large mtDNA deletion, and gene and protein expression.
    RESULTS: Our results revealed that 53.2% of the tumors harbor a mutation in at least one of the targeted susceptibility genes, and 16.9% harbor complementary mitochondrial mutations. More than 50% of the mitochondrial mutations were novel and predicted pathogenic, affecting mitochondrial oxidative phosphorylation. Large deletions were found in 26% of tumors, and depletion of mtDNA occurred in more than 87% of PCCs/PGLs. The reduction of the mitochondrial number was accompanied by a reduced expression of the regulators that promote mitochondrial biogenesis (PCG1α, NRF1, and TFAM). Further, P62 and LC3a gene expression suggested increased mitophagy, which is linked to mitochondrial dysfunction.
    CONCLUSION: The pathogenic role of these finding remains to be shown, but we suggest a complementarity and a potential contributing role in PCCs/PGLs tumorigenesis.
    Keywords:  genetic alterations; mitochondrial DNA; pheochromocytomas and paragangliomas
    DOI:  https://doi.org/10.3390/cancers14020269
  24. Free Radic Biol Med. 2022 Jan 16. pii: S0891-5849(22)00023-5. [Epub ahead of print]180 95-107
      Ferroptosis is a regulated form of cell death induced by iron (Fe)-dependent lipid peroxidation. At present, the underlying molecular mechanisms remain elusive. Herein, we hypothesized that mitochondria and the NRF2 (transcription factor nuclear factor E2-related factor 2) are potential mediators of ferroptosis, considering their well-established involvement in the oxidative stress pathway. We found that a high iron diet increased hepatic iron content and promoted glutathione (GSH) depletion, lipid peroxidation and oxidative stress. Dietary iron overload also decreased mRNA and protein expression levels of glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11), and increased mRNA and protein expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which are all markers of ferroptosis. Consistent with ferroptosis, iron overload promoted lipid peroxidation and the generation of mitochondrial reactive oxygen species (ROS), and decreased the mitochondrial membrane potential (MMP). Pre-treatment with deferoxamine mesylate (DFO, an iron chelator) alleviated ROS generation and lipid peroxidation, indicating a causative link between iron overload and lipid peroxidation. Suppression of mitochondrial oxidative stress attenuated ferroptosis. Experiments with HEK293T cells revealed that Fe-induced ferroptosis involved direct inhibition of NRF2 binding to antioxidant response elements (AREs) within the promoters of the gpx4 and slc7a11 genes, which in turn induced transcriptional silencing. In conclusion, our study provided a direct link between mitochondrial oxidative stress and ferroptosis via the NRF2-ARE pathway.
    Keywords:  Ferroptosis; Iron overload; Mitochondrial dysfunction; Oxidative stress; Vertebrates
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.01.012
  25. Redox Biol. 2022 Jan 15. pii: S2213-2317(22)00009-X. [Epub ahead of print]50 102237
      Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.
    Keywords:  Colitis; Inflammatory bowel disease; Macrophage; NAD(+); NAMPT; Phagocytic activity
    DOI:  https://doi.org/10.1016/j.redox.2022.102237
  26. Antioxid Redox Signal. 2022 Jan 19.
      AIMS: Impaired fatty acid oxidation (FAO) in mitochondria of hepatocytes causes lipid accumulation and excessive production of ROS and oxidative damage, as a result leads to nonalcoholic fatty liver disease (NAFLD). Fatty acid translocase (FAT/CD36), a transmembrane protein that facilitates the uptake of long-chain fatty acids (LCFAs), is recently found to be involved in FAO. The function of FAT/CD36 is associated with its subcellular localization. Palmitoylation, one of the most common lipid modifications, is generally thought to regulate FAT/CD36 subcellular localization. Here, we aimed to investigate the role of palmitoylation in FAT/CD36 localization to mitochondria and its influence on FAO in hepatocytes.RESULTS: We demonstrated that FAT/CD36 exists on the mitochondria of hepatocytes. Palmitoylation of FAT/CD36 was significantly up-regulated in NAFLD. Inhibition of FAT/CD36 palmitoylation resulted in an obvious increase in the distribution of FAT/CD36 to mitochondria of hepatocytes. Depalmitoylated FAT/CD36 on the mitochondrial membrane continues functioning by facilitating fatty acid trafficking to mitochondria. Abundant mitochondrial FAT/CD36 interacted with long-chain acyl-CoA synthetase 1 (ACSL1), and thus, more LCFAs were transported to ACSL1. This led to an increase in the generation of long-chain acyl-CoA, contributing to the enhancement of FAO and alleviating NAFLD. Innovation and Conclusion: This work revealed that inhibiting FAT/CD36 palmitoylation alleviates NAFLD by promoting FAT/CD36 localization to the mitochondria of hepatocytes. Mitochondrial FAT/CD36 functions as a molecular bridge between LCFAs and ACSL1 to increase the production of long-chain acyl-CoA thus promoting FAO, thereby avoiding lipid accumulation and over-production of ROS in hepatocytes.
    DOI:  https://doi.org/10.1089/ars.2021.0157
  27. Commun Biol. 2022 Jan 20. 5(1): 72
      Early diagnosis of acute myeloid leukemia (AML) in the pre-leukemic stage remains a clinical challenge, as pre-leukemic patients show no symptoms, lacking any known morphological or numerical abnormalities in blood cells. Here, we demonstrate that platelets with structurally abnormal mitochondria emerge at the pre-leukemic phase of AML, preceding detectable changes in blood cell counts or detection of leukemic blasts in blood. We visualized frozen-hydrated platelets from mice at different time points during AML development in situ using electron cryo-tomography (cryo-ET) and identified intracellular organelles through an unbiased semi-automatic process followed by quantitative measurement. A large proportion of platelets exhibited changes in the overall shape and depletion of organelles in AML. Notably, 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae, accompanied by a significant drop in ATP levels and altered expression of metabolism-related gene signatures. Our study demonstrates that detectable structural changes in pre-leukemic platelets may serve as a biomarker for the early diagnosis of AML.
    DOI:  https://doi.org/10.1038/s42003-022-03009-4
  28. Cancers (Basel). 2022 Jan 17. pii: 453. [Epub ahead of print]14(2):
      Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.
    Keywords:  FLT3-ITD; acute myeloid leukemia; autophagy; leukemic initiating cells; persistence; tyrosine kinase inhibitors
    DOI:  https://doi.org/10.3390/cancers14020453
  29. Oxid Med Cell Longev. 2022 ;2022 9151169
      Introduction: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a multifactorial intestinal disorder but its precise etiology remains elusive. As the cells of the intestinal mucosa have high energy demands, mitochondria may play a role in IBD pathogenesis. The present study is aimed at evaluating the expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes in IBD. Material and Methods. 286 intestinal biopsy samples from the terminal ileum, ascending colon, and rectum from 124 probands (34 CD, 33 UC, and 57 controls) were stained immunohistochemically for all five OXPHOS complexes and the voltage-dependent anion-selective channel 1 protein (VDAC1 or porin). Expression levels were compared in multivariate models including disease stage (CD and UC compared to controls) and age (pediatric/adult).Results: Analysis of the terminal ileum of CD patients revealed a significant reduction of complex II compared to controls, and a trend to lower levels was evident for VDAC1 and the other OXPHOS complexes except complex III. A similar pattern was found in the rectum of UC patients: VDAC1, complex I, complex II, and complex IV were all significantly reduced, and complex III and V showed a trend to lower levels. Reductions were more prominent in older patients compared to pediatric patients and more marked in UC than CD.
    Conclusion: A reduced mitochondrial mass is present in UC and CD compared to controls. This is potentially a result of alterations of mitochondrial biogenesis or mitophagy. Reductions were more pronounced in older patients compared to pediatric patients, and more prominent in UC than CD. Complex I and II are more severely compromised than the other OXPHOS complexes. This has potential therapeutic implications, since treatments boosting biogenesis or influencing mitophagy could be beneficial for IBD treatment. Additionally, substances specifically stimulating complex I activity should be tested in IBD treatment.
    DOI:  https://doi.org/10.1155/2022/9151169
  30. Cell Death Dis. 2022 Jan 18. 13(1): 63
      Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.
    DOI:  https://doi.org/10.1038/s41419-021-04334-y
  31. Cancers (Basel). 2022 Jan 11. pii: 335. [Epub ahead of print]14(2):
      Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1-13C]pyruvate (HP-13C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP-13C-MRI and conventional proton (1H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant (kPL) between 13C-pyruvate and 13C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing kPL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset (p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP-13C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP-13C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.
    Keywords:  cancer metabolism; hyperpolarized 13C magnetic resonance imaging; monocarboxylate transporter; renal cell carcinoma
    DOI:  https://doi.org/10.3390/cancers14020335
  32. Nat Commun. 2022 Jan 19. 13(1): 386
      Disordered hepatic glucagon response contributes to hyperglycemia in diabetes. The regulators involved in glucagon response are less understood. This work aims to investigate the roles of mitochondrial β-oxidation enzyme HADHA and its downstream ketone bodies in hepatic glucagon response. Here we show that glucagon challenge impairs expression of HADHA. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice, while HADHA knockdown augmented glucagon response. Stable isotope tracing shows that HADHA promotes ketone body production via β-oxidation. The ketone body β-hydroxybutyrate (BHB) but not acetoacetate suppresses gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with Glu543 site to facilitate FOXO1 nuclear exclusion. In HFD-fed mice, HADHA overexpression improved metabolic disorders, and these effects are abrogated by knockdown of BHB-producing enzyme. In conclusion, BHB is responsible for the inhibitory effect of HADHA on hepatic glucagon response, suggesting that HADHA activation or BHB elevation by pharmacological intervention hold promise in treating diabetes.
    DOI:  https://doi.org/10.1038/s41467-022-28044-x
  33. Nat Commun. 2022 Jan 19. 13(1): 385
      Mapping cell types across a tissue is a central concern of spatial biology, but cell type abundance is difficult to extract from spatial gene expression data. We introduce SpatialDecon, an algorithm for quantifying cell populations defined by single cell sequencing within the regions of spatial gene expression studies. SpatialDecon incorporates several advancements in gene expression deconvolution. We propose an algorithm harnessing log-normal regression and modelling background, outperforming classical least-squares methods. We compile cell profile matrices for 75 tissue types. We identify genes whose minimal expression by cancer cells makes them suitable for immune deconvolution in tumors. Using lung tumors, we create a dataset for benchmarking deconvolution methods against marker proteins. SpatialDecon is a simple and flexible tool for mapping cell types in spatial gene expression studies. It obtains cell abundance estimates that are spatially resolved, granular, and paired with highly multiplexed gene expression data.
    DOI:  https://doi.org/10.1038/s41467-022-28020-5
  34. FEBS J. 2022 Jan 20.
      Senescence is a multi-functional cell fate, characterized by an irreversible cell-cycle arrest and a pro-inflammatory phenotype, commonly known as the Senescence-Associated secretory Phenotype (SASP). Emerging evidence indicates that accumulation of senescent cells in multiple tissues, drives tissue dysfunction and several age-related conditions. This has spurred the academic community and industry to identify new therapeutic interventions targeting this process. Mitochondrial dysfunction is an often-unappreciated hallmark of cellular senescence which plays important roles not only in the senescence growth arrest but also in the development of the SASP and resistance to cell-death. Here, we review the evidence that supports a role for mitochondria in the development of senescence and describe the underlying mechanisms. Finally, we propose that a detailed road map of mitochondrial biology in senescence will be crucial to guide the future development of senotherapies.
    Keywords:  Mitochondria; SASP; aging; senescence
    DOI:  https://doi.org/10.1111/febs.16361
  35. Cells. 2022 Jan 07. pii: 197. [Epub ahead of print]11(2):
      Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H+ transporter activated by NH3 and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11's function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some "glutamine-addicted" cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.
    Keywords:  MCT4; ammonia; corneal endothelial dystrophy; glutamine; lactate
    DOI:  https://doi.org/10.3390/cells11020197
  36. Mol Cancer Res. 2022 Jan 19. pii: molcanres.0699.2021. [Epub ahead of print]
      Acidification is recognized as the predominant characteristic of the tumor microenvironment (TME) and contributes to tumor progression. However, the mechanism of extracellular acidic TME directly influences intercellular pathological responses remains unclear. Meanwhile, acidic TME is mainly ascribed to aberrant metabolism of lipids and glucose, but whether and how acidity affects metabolic reprogramming, especially for lipid metabolism, is still unknown. We found that lipid was significantly accumulated in liver cancer cells when exposed to acidic TME. Moreover, proteomic analysis showed that differentially expressed proteins were mainly clustered into fatty acid pathways. Subsequently, we found that acidification increased the expression of SCD1 by activating PI3K/AKT signaling pathway. Interestingly, we found that SCD1 directly bound to PPARα in the acidic TME, which vanished after 2-day reverse incubation in pH7.4 medium, implying extracellular acidosis might influence intercellular function by mediating the binding affinity between SCD1 and PPARα under different pH gradients. In summary, our data revealed that acidosis could significantly trigger fatty acid synthesis to promote liver tumorigenesis by upregulating SCD1 in a PI3K/AKT activation dependent manner and simultaneously promote SCD1 binding to PPARα. Our study not only provides direct mechanistic evidence to support the vital role of acidosis in lipid metabolic reprogramming, but also provides novel insights for determining the binding affinity of functional proteins as a molecular mechanism to better understand the role of the acidic TME in tumor development. Implications: The acidic TME contributes to lipid accumulation in liver cancer by activating the PI3K/AKT signaling pathway and promoting SCD1-PPARα binding.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0699