bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒11‒06
25 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Mol Cell. 2022 Oct 31. pii: S1097-2765(22)00962-5. [Epub ahead of print]
      Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.
    Keywords:  anaplerosis; cancer; cancer metabolism; metabolism; mitochondrion; redox; redox transfer; respiration
  2. EMBO Mol Med. 2022 Nov 02. e16082
      Metformin, a well-known antidiabetic drug, has been repurposed for cancer treatment; however, recently observed drug resistance and tumor metastasis have questioned its further application. Here, we found that long-term metformin exposure led to metabolic adaptation of hepatocellular carcinoma (HCC) cells, which was characterized by an obvious epithelial-mesenchymal transition (EMT) phenotype and compensatory elevation of oxidative phosphorylation (OXPHOS). TOMM34, a translocase of the outer mitochondrial membrane, was upregulated to promote tumor metastasis in response to metformin-induced metabolic stress. Mechanistically, TOMM34 interacted with ATP5B to preserve F1 FO -ATPase activity, which conferred mitochondrial OXPHOS and ATP production. This metabolic preference for OXPHOS suggested a large requirement of energy supply by cancer cells to survive and spread in response to therapeutic stress. Notably, disturbing the interaction between TOMM34 and ATP5B using Gboxin, a specific OXPHOS inhibitor, increased sensitivity to metformin and suppressed tumor progression both in vitro and in vivo. Overall, this study demonstrates a molecular link of the TOMM34/ATP5B-ATP synthesis axis during metformin adaptation and provides promising therapeutic targets for metformin sensitization in cancer treatment.
    Keywords:  TOMM34; hepatocellular carcinoma; metastasis; metformin adaptation; oxidative phosphorylation
  3. Nat Commun. 2022 Nov 04. 13(1): 6661
      Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis with cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA content and mitochondrial function are increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated NAD(P)H quinone dehydrogenase 1 (Nqo1). Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mitochondrial DNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.
  4. EMBO J. 2022 Oct 31. e111550
      Phosphoglycerate dehydrogenase (PHGDH) is a key serine biosynthesis enzyme whose aberrant expression promotes various types of tumors. Recently, PHGDH has been found to have some non-canonical functions beyond serine biosynthesis, but its specific mechanisms in tumorigenesis remain unclear. Here, we show that PHGDH localizes to the inner mitochondrial membrane and promotes the translation of mitochondrial DNA (mtDNA)-encoded proteins in liver cancer cells. Mechanistically, we demonstrate that mitochondrial PHGDH directly interacts with adenine nucleotide translocase 2 (ANT2) and then recruits mitochondrial elongation factor G2 (mtEFG2) to promote mitochondrial ribosome recycling efficiency, thereby promoting mtDNA-encoded protein expression and subsequent mitochondrial respiration. Moreover, we show that treatment with a mitochondrial translation inhibitor or depletion of mtEFG2 diminishes PHGDH-mediated tumor growth. Collectively, our findings uncover a previously unappreciated function of PHGDH in tumorigenesis acting via promotion of mitochondrial translation and bioenergetics.
    Keywords:  ANT2; PHGDH; liver cancer; mitochondrial translation; mtEFG2
  5. Mol Oncol. 2022 Nov 04.
      Mitochondrial DNA (mtDNA) somatic mutations play important roles in the initiation and progression of cancer. Although next-generation sequencing (NGS) of paired tumor and control samples has become a common practice to identify tumor-specific mtDNA mutations, the unique nature of mtDNA and NGS-associated sequencing bias could cause false positive/negative somatic mutation calling. Additionally, there are clinical scenarios where matched control tissues are unavailable for comparison. Therefore, a novel approach for accurately identifying somatic mtDNA variants is greatly needed, particularly in the absence of matched controls. In this study, the ground truth mtDNA variants orthogonally validated by triple-paired tumor, adjacent non-tumor, and blood samples were used to develop mitoSomatic, a random-forest-based machine learning tool. We demonstrated that mitoSomatic achieved area under the curve (AUC) values over 0.99 for identifying somatic mtDNA variants without paired control in three tumor types. In addition, mitoSomatic was also applicable in non-tumor tissues such as adjacent non-tumor and blood samples, suggesting the flexibility of mitoSomatic's classification capability. Furthermore, analysis of triple-paired samples identified a small group of variants with uncertain somatic/germline origin, whereas application of mitoSomatic significantly facilitated the prediction of their possible source. Finally, a control-free evaluation of the public pan-cancer NGS dataset with mitoSomatic revealed a substantial number of variants that were probably misclassified by conventional tumor-control comparison, further emphasizing the usefulness of mitoSomatic in application. Taken together, our study demonstrates that mitoSomatic is valuable for accurately identifying somatic mtDNA variants in mtDNA NGS data without paired controls, applicable for both tumor and non-tumor tissues.
    Keywords:  machine learning; mitochondrial DNA; next-generation sequencing; somatic mutations
  6. Nat Metab. 2022 Nov 03.
      Iron is essential to many fundamental biological processes, but its cellular compartmentalization and concentration must be tightly controlled. Although iron overload can contribute to obesity-associated metabolic deterioration, the subcellular localization and accumulation of iron in adipose tissue macrophages is largely unknown. Here, we show that macrophage mitochondrial iron levels control systemic metabolism in male mice by altering adipocyte iron concentrations. Using various transgenic mouse models to manipulate the macrophage mitochondrial matrix iron content in an inducible fashion, we demonstrate that lowering macrophage mitochondrial matrix iron increases numbers of M2-like macrophages in adipose tissue, lowers iron levels in adipocytes, attenuates inflammation and protects from high-fat-diet-induced metabolic deterioration. Conversely, elevating macrophage mitochondrial matrix iron increases M1-like macrophages and iron levels in adipocytes, exacerbates inflammation and worsens high-fat-diet-induced metabolic dysfunction. These phenotypes are robustly reproduced by transplantation of a small amount of fat from transgenic to wild-type mice. Taken together, we identify macrophage mitochondrial iron levels as a crucial determinant of systemic metabolic homeostasis in mice.
  7. EMBO Rep. 2022 Nov 02. e54978
      Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.
    Keywords:  LETM1; TMBIM5 (MICS1); mitochondrial Ca2+-H+ exchanger; mitochondrial metabolism; permeability transition pore
  8. Contact (Thousand Oaks). 2022 Jan-Dec;5:5
      A fundamental role of membrane-bound organelles is the compartmentalization and organization of cellular processes. Mitochondria perform an immense number of metabolic chemical reactions and to efficiently regulate these, the organelle organizes its inner membrane into distinct morphological domains, including its characteristic cristae membranes. In recent years, a structural feature of increasing apparent importance is the inter-connection between the mitochondrial exterior and other organelles at membrane contact sites (MCSs). Mitochondria form MCSs with almost every other organelle in the cell, including the endoplasmic reticulum, lipid droplets, and lysosomes, to coordinate global cellular metabolism with mitochondrial metabolism. However, these MCSs not only facilitate the transport of metabolites between organelles, but also directly impinge on the physical shape and functional organization inside mitochondria. In this review, we highlight recent advances in our understanding of how physical connections between other organelles and mitochondria both directly and indirectly influence the internal architecture of mitochondria.
    Keywords:  Ca2+; cristae; endoplasmic reticulum; inner mitochondrial membrane; interorganelle (inter-organelle); lipid droplet; lysosome; mitochondrion (mitochondria); phospholipid
  9. Nat Commun. 2022 Nov 04. 13(1): 6634
      Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is an inner mitochondrial membrane (IMM) protein that is dispensable for mitochondrial division yet essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in a fatal, adult-onset dilated cardiomyopathy accompanied by extensive mitochondrial and cardiac remodeling during the transition to heart failure. Prior to the onset of disease, knockout cardiac mitochondria displayed specific IMM defects: futile proton leak dependent upon the adenine nucleotide translocase and an increased sensitivity to the opening of the mitochondrial permeability transition pore, with which MTFP1 physically and genetically interacts. Collectively, our data reveal new functions of MTFP1 in the control of bioenergetic efficiency and cell death sensitivity and define its importance in preventing pathogenic cardiac remodeling.
  10. Cell Metab. 2022 Nov 01. pii: S1550-4131(22)00456-9. [Epub ahead of print]34(11): 1809-1823.e6
      Mitochondria have their own DNA (mtDNA), which is susceptible to the accumulation of disease-causing mutations. To prevent deleterious mutations from being inherited, the female germline has evolved a conserved quality control mechanism that remains poorly understood. Here, through a large-scale screen, we uncover a unique programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We find that PGM is developmentally triggered as germ cells enter meiosis by inhibition of the target of rapamycin complex 1 (TORC1). We identify a role for the RNA-binding protein Ataxin-2 (Atx2) in coordinating the timing of PGM with meiosis. We show that PGM requires the mitophagy receptor BNIP3, mitochondrial fission and translation factors, and members of the Atg1 complex, but not the mitophagy factors PINK1 and Parkin. Additionally, we report several factors that are critical for germline mtDNA quality control and show that pharmacological manipulation of one of these factors promotes mtDNA quality control.
    Keywords:  autophagy; germ line; germline; mitochondria; mitochondrial DNA; mitophagy; mtDNA; purifying selection; quality control
  11. Semin Cancer Biol. 2022 Oct 28. pii: S1044-579X(22)00204-8. [Epub ahead of print]86(Pt 3): 1216-1230
      Cancer cells undergo metabolic alterations to meet the immense demand for energy, building blocks, and redox potential. Tumors show glucose-avid and lactate-secreting behavior even in the presence of oxygen, a process known as aerobic glycolysis. Glycolysis is the backbone of cancer cell metabolism, and cancer cells have evolved various mechanisms to enhance it. Glucose metabolism is intertwined with other metabolic pathways, making cancer metabolism diverse and heterogeneous, where glycolysis plays a central role. Oncogenic signaling accelerates the metabolic activities of glycolytic enzymes, mainly by enhancing their expression or by post-translational modifications. Aerobic glycolysis ferments glucose into lactate which supports tumor growth and metastasis by various mechanisms. Herein, we focused on tumor glycolysis, especially its interactions with the pentose phosphate pathway, glutamine metabolism, one-carbon metabolism, and mitochondrial oxidation. Further, we describe the role and regulation of key glycolytic enzymes in cancer. We summarize the role of lactate, an end product of glycolysis, in tumor growth, and the metabolic adaptations during metastasis. Lastly, we briefly discuss limitations and future directions to improve our understanding of glucose metabolism in cancer.
    Keywords:  Cancer metabolism; Lactate; Metastasis; Tumor glycolysis; metabolic adaptations
  12. Sci Rep. 2022 Nov 02. 12(1): 18455
      There is a complete lack of highly sensitive and specific biomarkers for early pancreatic ductal adenocarcinoma (PDAC) diagnosis, limiting multi-modal therapeutic options. Mitochondrial DNA (mtDNA) is an excellent resource for biomarker discovery because of its high copy number and increased mutational frequency in cancer cells. We examined if mtDNA mutations can be detected in circulating extracellular vesicles (EVs) of PDAC patients and used for discerning between cancer and non-cancer subjects. A greater yield of circulating EVs (~ 1.4 fold; p = 0.002) was obtained in PDAC patients (n = 20) than non-cancer (NC) individuals (n = 10). PDAC-EVs contained a higher quantity of total DNA (~ 5.5 folds; p = 0.0001) than NC-EVs and had greater enrichment of mtDNA (~ 14.02-fold; p = 0.0001). PDAC-EVs also had higher levels of cardiolipin (a mitochondrial inner-membrane phospholipid), suggestive of their mitochondrial origin. All mtDNA mutations in PDAC-EVs were unique and frequency was remarkably higher. Most mtDNA mutations (41.5%) in PDAC-EVs were in the respiratory complex-I (RCI) (ND1-ND6), followed by the RCIII gene (CYTB; 11.2%). Among the non-coding genes, D-Loop and RNR2 exhibited the most mutations (15.2% each). Altogether, our study establishes, for the first time, that mtDNA mutations can be detected in circulating EVs and potentially serve as a tool for reliable PDAC diagnosis.
  13. Cell Metab. 2022 Nov 01. pii: S1550-4131(22)00459-4. [Epub ahead of print]34(11): 1620-1653
      The analogy of mitochondria as powerhouses has expired. Mitochondria are living, dynamic, maternally inherited, energy-transforming, biosynthetic, and signaling organelles that actively transduce biological information. We argue that mitochondria are the processor of the cell, and together with the nucleus and other organelles they constitute the mitochondrial information processing system (MIPS). In a three-step process, mitochondria (1) sense and respond to both endogenous and environmental inputs through morphological and functional remodeling; (2) integrate information through dynamic, network-based physical interactions and diffusion mechanisms; and (3) produce output signals that tune the functions of other organelles and systemically regulate physiology. This input-to-output transformation allows mitochondria to transduce metabolic, biochemical, neuroendocrine, and other local or systemic signals that enhance organismal adaptation. An explicit focus on mitochondrial signal transduction emphasizes the role of communication in mitochondrial biology. This framework also opens new avenues to understand how mitochondria mediate inter-organ processes underlying human health.
    Keywords:  amplification; communication; energy; evolution; health; membrane potential; metabokines; mito-nuclear signaling; mitochondrial networks; mitokines; mitotypes; receptors; signal transduction; steroid hormones; stress responses; tissue-specific
  14. J Physiol. 2022 Nov 03.
      KEY POINTS: Estrogen loss in women increases risk for cardiometabolic diseases, a link that has been partially attributed to negative impacts on mitochondria and energy metabolism. To study the effect of oral combined contraceptives (OCs) on hepatic and skeletal muscle mitochondria and whole-body energy metabolism, we used an animal model of OCs which mimics the human condition of cessation of hormonal cycling in the low estrogen, high progesterone state. OC treated mice have increased hepatic mitochondrial oxidative stress and decreased physical activity and energy expenditure, despite displaying lower adiposity and liver fat at this timepoint. These pre-clinical data reveal tissue-specific effects of OCs that likely underly the clinical findings of increased cardiometabolic disease in women who use OCs compared to non-users, when matched for obesity.ABSTRACT: Compared to age-matched men, pre-menopausal women show greater resilience against cardiovascular disease (CVD), hepatic steatosis, diabetes, and obesity - findings that are widely attributed to estrogen. However, meta-analysis data suggest that current use of oral combined contraceptives (OC) is a risk factor for myocardial infarction, and OC use further compounds with metabolic disease risk factors to increase CVD susceptibility. While mitochondrial function in tissues such as the liver and skeletal muscle is an emerging mechanism by which estrogen may confer its protection, effects of OC use on mitochondria and metabolism in the context of disease risk remains unexplored. To answer this question, female C57Bl/6J mice were fed a high fat diet and treated with vehicle or OCs for 3, 12, or 20 weeks (n = 6-12 per group) at a dose and ratio that mimics the human condition of cycle cessation in the low estrogen, high progesterone stage. Liver and skeletal muscle mitochondrial function (respiratory capacity, H2 O2 , coupling) was measured along with clinical outcomes of cardiometabolic disease such as obesity, glucose tolerance, hepatic steatosis, and aortic atherosclerosis. Main findings indicate that regardless of treatment duration, OCs robustly increase hepatic mitochondrial H2 O2 levels, likely due to diminished antioxidant capacity, but has no impact on muscle mitochondrial H2 O2 . Furthermore, OC treated mice had lower adiposity and hepatic triglyceride content compared to control mice despite reduced wheel running, spontaneous physical activity, and total energy expenditure. Together, these studies describe tissue-specific effects of OC use on mitochondria as well as variable impacts on markers of metabolic disease susceptibility. Abstract figure legend This article is protected by copyright. All rights reserved.
    Keywords:  birth control; estrogen; mitochondria; physical activity
  15. Autophagy. 2022 Oct 31.
      Macroautophagy/autophagy is a highly conserved catabolic process pivotal to cellular homeostasis and support of tumorigenesis. Being a potential therapeutic target for cancer, we have worked to understand the implications of autophagy inhibition both systemically, and tumor-specifically. We utilized inducible expression of Atg5 shRNA to temporally control autophagy levels in a reversible manner to study the effects of tumor-intrinsic and systemic autophagic loss and restoration on established KrasG12D/+;trp53-/- (KP) lung tumor growth. We reported that transient systemic ATG5 loss significantly reduces KP lung tumor growth. Through in vivo isotope tracing and metabolic flux analyses, we noted that systemic ATG5 knockdown significantly reduces the uptake of glucose and lactate in lung tumors, leading to impaired TCA cycle metabolism and biosynthesis. Additionally, we observed an increased tumor T cell infiltration in the absence of systemic ATG5, which is essential for T cell-mediated tumor killing. Moreover, the impaired tumor metabolism and increased T cell infiltration are sustained when autophagy is restored in a short term. Finally, we found that intermittent systemic ATG5 knockdown, a mock therapy situation, significantly prolongs the lifespan of mice bearing KP lung tumors. Our findings lay the proof of concept for inhibition of autophagy as a valid approach to cancer therapy.
    Keywords:  KRAS; autophagy; cancer metabolism; cancer therapy; immune evasion; lung tumor
  16. Nat Commun. 2022 Nov 04. 13(1): 6622
      Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.
  17. Cancer Res. 2022 Nov 01. pii: CAN-22-1029. [Epub ahead of print]
      The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the nicotinamide adenine dinucleotide (NAD)+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate (α-KG)/2- hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and β-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and β-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma.
  18. Sci Rep. 2022 Nov 03. 12(1): 18561
      Raman spectroscopy and Raman imaging were used to identify the biochemical and structural features of human cancer lung cells (CCL-185) and the cancer cells supplemented with glucose and deuterated glucose at normal and hyperglycemia conditions. We found that isotope substitution of glucose by deuterated glucose allows to separate de novo lipid synthesis from exogenous uptake of lipids obtained from the diet. We demonstrated that glucose is largely utilized for de novo lipid synthesis. Our results provide a direct evidence that high level of glucose decreases the metabolism via oxidative phosphorylation in mitochondria in cancer cells and shifts the metabolism to glycolysis via Warburg effect. It suggests that hyperglycemia is a factor that may contribute to a more malignant phenotype of cancer cells by inhibition of oxidative phosphorylation and apoptosis.
  19. mBio. 2022 Oct 31. e0218722
      Oxygenic photoautotrophic bacteria, cyanobacteria, have the tricarboxylic acid (TCA) cycle, and metabolite production using the cyanobacterial TCA cycle has been spotlighted recently. The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) has been used in various studies on the cyanobacterial TCA cycle. Malate oxidation in the TCA cycle is generally catalyzed by malate dehydrogenase (MDH). However, Synechocystis 6803 MDH (SyMDH) is less active than MDHs from other organisms. Additionally, SyMDH uses only NAD+ as a coenzyme, unlike other TCA cycle enzymes from Synechocystis 6803 that use NADP+. These results suggest that MDH rarely catalyzes malate oxidation in the cyanobacterial TCA cycle. Another enzyme catalyzing malate oxidation is malic enzyme (ME). We clarified which enzyme oxidizes malate that originates from the cyanobacterial TCA cycle using analyses focusing on ME and MDH. In contrast to SyMDH, Synechocystis 6803 ME (SyME) showed high activity when NADP+ was used as a coenzyme. Unlike the Synechocystis 6803 mutant lacking SyMDH, the mutant lacking SyME accumulated malate in the cells. ME was more highly preserved in the cyanobacterial genomes than MDH. These results indicate that ME mainly oxidizes malate that originates from the cyanobacterial TCA cycle (named the ME-dependent TCA cycle). The ME-dependent TCA cycle generates NADPH, not NADH. This is consistent with previous reports that NADPH is an electron carrier in the cyanobacterial respiratory chain. Our finding suggests the diversity of enzymes involved in the TCA cycle in the organisms, and analyses such as those performed in this study are necessary to determine the enzymes. IMPORTANCE Oxygenic photoautotrophic bacteria, namely, cyanobacteria, have the tricarboxylic acid (TCA) cycle. Recently, metabolite production using the cyanobacterial TCA cycle has been well studied. To enhance the production volume of metabolites, understanding the biochemical properties of the cyanobacterial TCA cycle is required. Generally, malate dehydrogenase oxidizes malate in the TCA cycle. However, cyanobacterial malate dehydrogenase shows low activity and does not use NADP+ as a coenzyme, unlike other cyanobacterial TCA cycle enzymes. Our analyses revealed that another malate oxidation enzyme, the malic enzyme, mainly oxidizes malate that originates from the cyanobacterial TCA cycle. These findings provide better insights into metabolite production using the cyanobacterial TCA cycle. Furthermore, our findings suggest that the enzymes related to the TCA cycle vary from organism to organism and emphasize the importance of analyses to identify the enzymes such as those performed in this study.
    Keywords:  aerobic respiration; cyanobacteria; malate dehydrogenase; malic enzyme; tricarboxylic acid cycle
  20. Sci Rep. 2022 Nov 04. 12(1): 18687
      Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C →  T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.
  21. Leukemia. 2022 Nov 04.
      Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.
  22. J Neuromuscul Dis. 2022 Oct 28.
      BACKGROUND: The number of mutations in nuclear encoded genes causing mitochondrial disease is ever increasing. Identification of these mutations is particularly important in the diagnosis of neuromuscular disorders as their presentation may mimic other acquired disorders.We present a novel heterozygous variant in mitochondrial fission factor (MFF) which mimics myasthenia gravis.OBJECTIVE: To determine if the MFF c.937G>A, p.E313K variant causes a mild mitochondrial phenotype.
    METHODS: We used whole exome sequencing (WES) to identify a novel heterozygous variant in MFF in a patient with ptosis, fatigue and muscle weakness. Using patient derived fibroblasts, we performed assays to evaluate mitochondrial and peroxisome dynamics.
    RESULTS: We show that fibroblasts derived from this patient are defective in mitochondrial fission, despite normal recruitment of Drp1 to the mitochondria.
    CONCLUSIONS: The MFF c.937G>A, p.E313K variant leads to a mild mitochondrial phenotype and is associated with defective mitochondrial fission in patient-derived fibroblasts.
    Keywords:  MFF; Mitochondrial myopathies; mitochondrial dynamics; mitochondrial fission factor
  23. Mol Syst Biol. 2022 11;18(11): e11033
      Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan-cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13 C-flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial-mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.
    Keywords:  cancer metabolism; cell lines; metabolic flux; metabolomics; omics
  24. Drug Resist Updat. 2022 Oct 28. pii: S1368-7646(22)00087-5. [Epub ahead of print]65 100888
      Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the predominant histological subtype. Despite the emergence of targeted and immune-based therapies that have considerably improved the clinical outcomes of selected patients, the overall NSCLC survival rate remains poor. NSCLC patients experience clinical relapse mainly because of chemoresistance. One promising therapeutic approach is targeting specific molecular vulnerabilities that are associated with the metabolic reprogramming of cancer cells. This strategy relies on evidence that cancer cells rewire their metabolism to sustain their uncontrolled growth as well as invasive and metastatic properties, promoting adaptive resistance to chemo-radiotherapy. A critical component of this malignant transformation is the increased dependency on high levels of heat shock proteins (HSPs), which support the elevated protein folding demand and quality control of misfolded oncoproteins. Here, we provide an overview of the literature on metabolism reprogramming, deregulation of mitochondrion and on the role of HSPs in promoting malignancy in lung and other cancer types. A particular focus is dedicated to the role of mitochondrial HSP60 (HSPD1) in NSCLC metabolism and drug resistance for the potential development of new resistance-defying anti-HSP drugs.
    Keywords:  Cancer; Drug resistance; HSP60; Heat shock proteins; Metabolism; Non-small cell lung cancer
  25. Mol Syst Biol. 2022 11;18(11): e11006
      The unravelling of the complexity of cellular metabolism is in its infancy. Cancer-associated genetic alterations may result in changes to cellular metabolism that aid in understanding phenotypic changes, reveal detectable metabolic signatures, or elucidate vulnerabilities to particular drugs. To understand cancer-associated metabolic transformation, we performed untargeted metabolite analysis of 173 different cancer cell lines from 11 different tissues under constant conditions for 1,099 different species using mass spectrometry (MS). We correlate known cancer-associated mutations and gene expression programs with metabolic signatures, generating novel associations of known metabolic pathways with known cancer drivers. We show that metabolic activity correlates with drug sensitivity and use metabolic activity to predict drug response and synergy. Finally, we study the metabolic heterogeneity of cancer mutations across tissues, and find that genes exhibit a range of context specific, and more general metabolic control.
    Keywords:  cancer; heterogeneity; metabolomics; mutation