bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2023‒02‒05
28 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. PNAS Nexus. 2022 Nov;1(5): pgac276
      Respiratory complex I [NADH:ubiquinone (UQ) oxidoreductase] captures the free energy released from NADH oxidation and UQ reduction to pump four protons across an energy-transducing membrane and power ATP synthesis. Mechanisms for long-range energy coupling in complex I have been proposed from structural data but not yet evaluated by robust biophysical and biochemical analyses. Here, we use the powerful bacterial model system Paracoccus denitrificans to investigate 14 mutations of key residues in the membrane-domain Nqo13/ND4 subunit, defining the rates and reversibility of catalysis and the number of protons pumped per NADH oxidized. We reveal new insights into the roles of highly conserved charged residues in lateral energy transduction, confirm the purely structural role of the Nqo12/ND5 transverse helix, and evaluate a proposed hydrated channel for proton uptake. Importantly, even when catalysis is compromised the enzyme remains strictly coupled (four protons are pumped per NADH oxidized), providing no evidence for escape cycles that circumvent blocked proton-pumping steps.
    Keywords:  NADH:ubiquinone oxidoreductase; biological energy transduction; electron transport chain; proton pumping; respiratory chain
  2. Cancer Biol Ther. 2023 Dec 31. 24(1): 2170669
      In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were "Metabolic Reprogramming in Cancer" and "Glycolysis Activation in Cancer (Warburg Effect)." We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by high-resolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial-lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC.
    Keywords:  HIF1a; MIRCAF2; MIRCAF3; MISTR2; MISTR3; NDUFA4; NDUFA4L2; NPC2; Warburg effect; ccRCC; co-immunofluorescence; expansion microscopy; glycolysis; hypoxia; kidney cancer; lysosome; mass spectrometry; mitochondria; mitochondrial respiration; oxidative phosphorylation
  3. Nature. 2023 Feb 01.
      Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.
  4. Curr Urol. 2022 Dec;16(4): 207-212
      Mitochondria are more than just the cellular powerhouse. They also play key roles in vital functions such as apoptosis, metabolism regulation, and other intracellular interactions. The mitochondrial DNA (mtDNA) encodes for 12 subunits of the oxidative phosphorylation (OXPHOS) system. Depletion of mtDNA in androgen-dependent prostate cancer (PCa) cell lines renders them androgen-independent and more aggressive. Paradoxically, pharmaceutical inhibition of OXPHOS is lethal for subsets of PCa cells, whereas others become dependent on androgen receptor (AR) signaling for survival. Given that the AR-mitochondria interaction is critical for early PCa, it is crucial to understand the details of this interaction. Technical hurdles have made mitochondria traditionally difficult to study, with many techniques used for isolation masking the properties of given individual mitochondria. Although the isolation of mitochondria enables us to study OXPHOS, we miss the context in which mitochondria interact with the rest of the cell. Both AR signaling and mtDNA affect apoptosis, metabolism regulation, cellular calcium storage and homeostasis, intracellular calcium signaling, and redox homeostasis. In this review, we will attempt to understand how the crosstalk between AR-mtDNA-OXPHOS is responsible for "life or death" decisions inside the cells. Our aim is to point toward potential vulnerabilities that can lead to the discovery of novel therapeutic targets.
    Keywords:  Androgen receptor; Mitochondria; Oxidative phosphorylation; Prostate cancer
  5. bioRxiv. 2023 Jan 20. pii: 2023.01.19.524841. [Epub ahead of print]
      Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs' intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (Δ Ψm ), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics.Author Summary: Pneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.
  6. Autophagy. 2023 Feb 01. 1-3
      Age-related human pathologies present with a multitude of molecular and metabolic phenotypes, which individually or synergistically contribute to tissue degeneration. However, current lack of understanding of the interdependence of these molecular pathologies limits the potential range of existing therapeutic intervention strategies. In our study, we set out to understand the chain of molecular events, which underlie the loss of cellular viability in macroautophagy/autophagy deficiency associated with aging and age-related disease. We discover a novel axis linking autophagy, a cellular waste disposal pathway, and a metabolite, nicotinamide adenine dinucleotide (NAD). The axis connects multiple organelles, molecules and stress response pathways mediating cellular demise when autophagy becomes dysfunctional. By elucidating the steps on the path from efficient mitochondrial recycling to NAD maintenance and ultimately cell viability, we highlight targets potentially receptive to therapeutic interventions in a range of genetic and age-related diseases associated with autophagy dysfunction.Abbreviations: IMM: inner mitochondrial membrane; NAD: nicotinamide dinucleotide; OXPHOS: oxidative phosphorylation; PARP: poly(ADP-ribose) polymerase; ROS: reactive oxygen species.
    Keywords:  Aging; DNA damage; NAD; PARP; ROS; autophagy; mitochondria; mitophagy; sirtuins
  7. Sci Adv. 2023 Feb 03. 9(5): eade8701
      Macrophage metabolic plasticity enables repurposing of electron transport from energy generation to inflammation and host defense. Altered respiratory complex II function has been implicated in cancer, diabetes, and inflammation, but regulatory mechanisms are incompletely understood. Here, we show that macrophage inflammatory activation triggers Complex II disassembly and succinate dehydrogenase subunit B loss through sequestration and selective mitophagy. Mitochondrial fission supported lipopolysaccharide-stimulated succinate dehydrogenase subunit B degradation but not sequestration. We hypothesized that this Complex II regulatory mechanism might be coordinated by the mitochondrial phospholipid cardiolipin. Cardiolipin synthase knockdown prevented lipopolysaccharide-induced metabolic remodeling and Complex II disassembly, sequestration, and degradation. Cardiolipin-depleted macrophages were defective in lipopolysaccharide-induced pro-inflammatory cytokine production, a phenotype partially rescued by Complex II inhibition. Thus, cardiolipin acts as a critical organizer of inflammatory metabolic remodeling.
  8. PNAS Nexus. 2022 Sep;1(4): pgac192
      Mitochondria are cellular organelles of crucial relevance for the survival of metazoan organisms. Damage to the mitochondrial DNA can give rise to a variety of mitochondrial diseases and is thought also to be involved in the aging process. The fate of mtDNA mutants is controlled by their synthesis as well as degradation and mathematical models can help to better understand this complex interplay. We present here a model that combines a replicative advantage for mtDNA mutants with selective degradation enabled by mitochondrial fission and fusion processes. The model not only shows that the cell has efficient means to deal with (many) types of mutants but, surprisingly, also predicts that under certain conditions a stable co-existence of mutant and wild-type mtDNAs is possible. We discuss how this new finding might explain how mitochondria can be at the heart of processes with such different phenotypes as mitochondrial diseases and aging.
    Keywords:  aging; mathematical model; mitochondrial disease
  9. bioRxiv. 2023 Jan 19. pii: 2023.01.19.524708. [Epub ahead of print]
      A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein we identify the molecular mechanisms involved, demonstrating that TRAP1: i) binds both mitochondrial and cytosolic ribosomes as well as translation elongation factors, ii) slows down translation elongation rate, and iii) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.
  10. Pharmacol Res. 2023 Jan 31. pii: S1043-6618(23)00039-7. [Epub ahead of print] 106683
      In spite of the huge advancements in both diagnosis and interventions, hormone refractory prostate cancer (HRPC) remains a major hurdle in prostate cancer (PCa). Metabolic reprogramming plays a key role in PCa oncogenesis and resistance. However, the dynamics between metabolism and oncogenesis are not fully understood. Here, we demonstrate that two multi-target natural products, cannabidiol (CBD) and cannabigerol (CBG), suppress HRPC development in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model by reprogramming metabolic and oncogenic signalling. Mechanistically, CBD increases glycolytic capacity and inhibits oxidative phosphorylation in enzalutamide-resistant HRPC cells. This action of CBD originates from its effect on metabolic plasticity via modulation of VDAC1 and hexokinase II (HKII) coupling on the outer mitochondrial membrane, which leads to strong shifts of mitochondrial functions and oncogenic signalling pathways. The effect of CBG on enzalutamide-resistant HRPC cells was less pronounced than CBD and only partially attributable to its action on mitochondria. However, when optimally combined, these two cannabinoids exhibited strong anti-tumor effects in TRAMP mice, even when these had become refractory to enzalutamide, thus pointing to their therapeutical potential against PCa.
  11. EMBO J. 2023 Feb 02. e112094
      DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.
    Keywords:  ANT2; ATM; DNA-PKcs; VDAC2; mitochondrial oxidative stress checkpoint
  12. Free Radic Biol Med. 2023 Jan 31. pii: S0891-5849(23)00043-6. [Epub ahead of print]
      Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5-80 μM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24 h. Leukemic cells produced more acidic organelles to tolerate 5-10 μM CQ. CQ (20-80 μM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80 μM CQ challenge, co-treatment with the inhibitor of F0 part of mitochondrial H+-ATP synthase, oligomycin (40 nM), prevented the elevation of oxidants as well as pHi, and attenuated stresses on mitochondria, cell survival, and cell proliferation. Besides, oligomycin-treated cells obviously displayed the lysosomal peripheralization and plasma membrane blebbing, suggesting that these cells were in process of lysosomal exocytosis and microvesicle release. Enhanced motion of these secretory processes allowed the cells to exclude CQ and repair necrotic injury. Together, the oxidant production and the proton dynamic interconnection among lysosomes, mitochondria, and cytosol are crucial for leukemic susceptibility to lysosomotropic chemotherapeutics.
    Keywords:  Intracellular pH; Lysosomal inhibition; Mitochondrial H(+)-ATP synthase; Multidrug resistance; Reactive oxygen/nitrogen species; Secretory process
  13. Front Oncol. 2022 ;12 1043670
      Background: Ovarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion.Methods: Using our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities.
    Results: Independent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids.
    Discussion: Our studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.
    Keywords:  adhesion; cycloheximide; mitobiogenesis; mitochondria; mitophagy; ovarian cancer metabolism; reoxygenation; spheroid
  14. Bioessays. 2023 Jan 29. e2200160
      Mitochondria hold diverse and pivotal roles in fundamental processes that govern cell survival, differentiation, and death, in addition to organismal growth, maintenance, and aging. The mitochondrial protein import system is a major contributor to mitochondrial biogenesis and lies at the crossroads between mitochondrial and cellular homeostasis. Recent findings highlight the mitochondrial protein import system as a signaling hub, receiving inputs from other cellular compartments and adjusting its function accordingly. Impairment of protein import, in a physiological, or disease context, elicits adaptive responses inside and outside mitochondria. In this review, we discuss recent developments, relevant to the mechanisms of mitochondrial protein import regulation, with a particular focus on quality control, proteostatic and metabolic cellular responses, triggered upon impairment of mitochondrial protein import.
    Keywords:  metabolism; mitochondrial protein import; mitochondrial unfolded protein response; mitophagy; proteostasis
  15. Redox Rep. 2023 Dec;28(1): 2168635
      BACKGROUND: Methionine sulfoxide reductases are found in all aerobic organisms. They function in antioxidant defense, cellular regulation by reversible oxidation of methionine in proteins, and in protein structure. However, very few in vivo binding partners or substrates of the reductases have been identified.METHODS: We implemented a proximity labeling method, TurboID, to covalently link mitochondrial methionine sulfoxide reductase A (MSRA) to its binding partners in HEK293 cells. Proteomic analyses were performed to identify putative binding partners.
    RESULTS: We show that human Ndufaf2, also called mimitin, is a binding partner of MSRA as well as all 3 MSRBs. We found that both methionine residues in Ndufaf2 were susceptible to oxidation by hydrogen peroxide and that the methionine sulfoxide reductases can reduce these methionine sulfoxide residues back to methionine.
    CONCLUSION: Methionine sulfoxide reductases can reduce methionine sulfoxide back to methionine in Ndufaf2. In addition to a repair function, it also creates a mechanism that could regulate cellular processes by modulation of methionine oxidation in Ndufaf2.
    Keywords:  Methionine sulfoxide reductase; Ndufaf2; TurboID proximity labeling; mass spectrometry; methionine oxidation; mimitin; mitochondrial complex I; oxidative stress
  16. bioRxiv. 2023 Jan 12. pii: 2023.01.11.523668. [Epub ahead of print]
      The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered but SHMT2- and serine dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis does not respond to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 as a major glycine-consuming enzyme.
  17. Genet Test Mol Biomarkers. 2023 Jan;27(1): 5-11
      Aims: Mitochondrial functional transformation contributes to the carcinogenesis of the prostate by meeting the metabolic needs of cancer cells. Mitochondrial transcription factor A (TFAM) is a pivotal regulator that maintains homeostasis of mitochondrial function. However, its role in prostate carcinogenesis has not been well elucidated. Materials and Methods: In the present study, we analyzed the expression of TFAM in normal prostate tissue and prostate cancer using public databases; a prostate-tissue chip was used to verify the results. The expression of TFAM in normal cells and in prostate cancer cells was determined by western blotting analysis. We knocked down TFAM in the prostate cancer cell line PC3 using a specific shRNA to explore the potential effects of TFAM in prostatic carcinogenesis. Results: We observed higher expression levels of TFAM in prostate cancer tissue than in normal prostate tissue and tumor adjacent normal tissues. A receiver operating characteristic curve was drawn that demonstrated the diagnostic efficacy of using TFAM expression for prostate cancer prognoses. Elevated levels of TFAM may indicate poorer overall survival in prostate cancer patients. Western blotting assays also showed that relative to the normal prostatic epithelial cell line RWPE-1, prostate cancer cell lines PC3 and DU145 expressed more TFAM protein. Furthermore, knockdown of TFAM inhibited the colony-formation capability of PC3 cells. Conclusion: Collectively, these results suggest that TFAM promotes carcinogenesis of the prostate, and may constitute a marker to be used in the diagnosis and prognosis of prostate cancer.
    Keywords:  TFAM; colony formation; prostate cancer; public database; tissue chip
  18. Methods Cell Biol. 2023 ;pii: S0091-679X(22)00143-1. [Epub ahead of print]174 93-111
      Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.
    Keywords:  Antimycin; Autophagy; CGAS/STING1; NLRP3 inflammasome; Oligomycin; PRKN; SARRP
  19. Nat Cancer. 2023 Feb 02.
      Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.
  20. J Cancer Res Clin Oncol. 2023 Jan 31.
      INTRODUCTION: Radiotherapy is a mainstay of cancer treatment. Clinical studies revealed a heterogenous response to radiotherapy, from a complete response to even disease progression. To that end, finding the relative prognostic factors of disease outcomes and predictive factors of treatment efficacy and toxicity is essential. It has been demonstrated that radiation response depends on DNA damage response, cell cycle phase, oxygen concentration, and growth rate. Emerging evidence suggests that altered mitochondrial metabolism is associated with radioresistance.METHODS: This article provides a comprehensive evaluation of the role of mitochondria in radiotherapy efficacy and toxicity. In addition, it demonstrates how mitochondria might be involved in the famous 6Rs of radiobiology.
    RESULTS: In terms of this idea, decreasing the mitochondrial metabolism of cancer cells may increase radiation response, and enhancing the mitochondrial metabolism of normal cells may reduce radiation toxicity. Enhancing the normal cells (including immune cells) mitochondrial metabolism can potentially improve the tumor response by enhancing immune reactivation. Future studies are invited to examine the impacts of mitochondrial metabolism on radiation efficacy and toxicity. Improving radiotherapy response with diminishing cancer cells' mitochondrial metabolism, and reducing radiotherapy toxicity with enhancing normal cells' mitochondrial metabolism.
    Keywords:  Mitochondria; Personalized oncology; Radiosensitivity; Radiotherapy
  21. BMC Cancer. 2023 Feb 03. 23(1): 117
      BACKGROUND: Recurrence due to the development of radioresistance remains a major challenge in the clinical management of nasopharyngeal carcinoma. The objective of this study was to increase the sensitivity of nasopharyngeal carcinoma cells to ionizing radiation by enhancing oxidative stress and ferroptosis caused by disrupting the mitochondrial anti-oxidant enzyme system.METHODS: Oxidative stress cell model was constructed by SOD2 knockdown using shRNA. The expression and activity of DHODH was suppressed by siRNA and brequinar in SOD2 depleted cells. Protein levels were determined by western blotting and ferroptosis was assessed by C11 BODIPY and malondialdehyde assay. Cell viability was evaluated using CCK-8 assay while radiotoxicity was assessed by colony formation assay. Cellular ATP level was determined by ATP assay kits, ROS was determined by DCFD and DHE, while mitochondrial oxygen consumption was determined by seahorse assay. Data were analyzed by two-tailed independent t-test.
    RESULTS: Radiation upregulated SOD2 expression and SOD2 depletion increased cellular O2.-, malondialdehyde, and the fluorescence intensity of oxidized C11 BODIPY. It also resulted in mitochondrial damage. Its depletion decreased colony formation both under ionizing and non-ionizing radiation conditions. The ferroptosis inhibitor, deferoxamine, rescued cell viability and colony formation in SOD2 depleted cells. Cellular level of malondialdehyde, fluorescence intensity of oxidized C11 BODIPY, O2.- level, ATP, and mitochondrial oxygen consumption decreased following DHODH inhibition in SOD2 depleted cells. Cell viability and colony formation was rescued by DHODH inhibition in SOD2 depleted cells.
    CONCLUSION: Inducing oxidative stress by SOD2 inhibition sensitized nasopharyngeal carcinoma cells to ionizing radiation via ferroptosis induction. This was found to be dependent on DHODH activity. This suggests that DHODH inhibitors should be used with caution during radiotherapy in nasopharyngeal carcinoma patients.
    Keywords:  DHODH; Ferroptosis; Nasopharyngeal carcinoma; Oxidative stress; SOD2
  22. medRxiv. 2023 Jan 19. pii: 2023.01.19.23284696. [Epub ahead of print]
      Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis -acting genetic control over mtDNA abundance and is itself under trans -acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.
  23. Res Sq. 2023 Jan 10. pii: [Epub ahead of print]
      For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.
  24. Cancer Res. 2023 Jan 30. pii: CAN-22-1844. [Epub ahead of print]
      Angiogenesis is vital for tumor growth and metastasis. Emerging evidence suggests that metabolic reprogramming in endothelial cells (ECs) may affect angiogenesis. Here, we showed that multiple regulators in the fructose metabolism pathway, especially fructose transporter SLC2A5 and fructose-metabolizing enzyme ketohexokinase (KHK), were upregulated in tumor endothelial cells from hepatocellular carcinoma (HCC). In mouse models with hepatoma xenografts or with Myc/sgp53-induced liver cancer, dietary fructose enhanced tumor angiogenesis, tumor growth, and metastasis, which could be attenuated by treatment with an inhibitor of SLC2A5. Furthermore, vessel growth was substantially increased in fructose-containing matrigel compared to PBS-matrigel. Inhibiting fructose metabolism in EC cells in vivo using EC-targeted nanoparticles loaded with siRNA against KHK significantly abolished fructose-induced tumor angiogenesis. Fructose treatment promoted the proliferation, migration and tube formation of ECs and stimulated mitochondrial respiration and ATP production. Elevated fructose metabolism activated AMPK to fuel mitochondrial respiration, resulting in enhanced EC migration. Fructose metabolism was increased under hypoxic conditions as a result of HIF1α-mediated upregulation of multiple genes in the fructose metabolism pathway. These findings highlight the significance of fructose metabolism in ECs for promoting tumor angiogenesis. Restricting fructose intake or targeting fructose metabolism is a potential strategy to reduce angiogenesis and suppress tumor growth.
  25. Cancer Cell. 2023 Feb 02. pii: S1535-6108(23)00003-X. [Epub ahead of print]
      Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.
    Keywords:  Angioimmunoblastic T cell lymphoma; Idh2; T follicular helper cells; Tet2; cytokines; epigenetics; germinal center B cells; preclinical mouse model; therapeutic agents; tumor microenvironment
  26. Proc Natl Acad Sci U S A. 2023 Feb 07. 120(6): e2212072120
      Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.
    Keywords:  ATM; DNA repair; KEAP1; drug resistance; redox metabolism
  27. Nature. 2023 Feb 01.
    Keywords:  Cancer; Metabolism
  28. bioRxiv. 2023 Jan 18. pii: 2023.01.16.524176. [Epub ahead of print]
      Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to the lack of native 3D views of cristae morphology. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with well-defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe elongated mitochondria with a notable stacking phenotype, as well as an absence of tubular cristae, when only l-Opa1 is present. In contrast, when mitochondria contain mainly s-Opa1, we observe irregular cristae packing, an increase in globular cristae, and decreased matrix condensation. Notably, we find the absence of l-Opa1 results in mitochondria with wider cristae junctions. BH3 profiling reveals that absence of l-Opa1 reduces cytochrome c release in response to pro-apoptotic stimuli and protects cells from apoptosis induced by anti-cancer agents. We discuss the implications Opa1-dependent cristae morphologies in cell death initiation.Highlights: In situ ultrastructural characterization of mitochondrial cristae with different forms of Opa1. Mitochondria with predominantly l-Opa1 show cristae stacking, longer cristae compared to WT, but also a reduction of globular cristae and no tubular cristae.Mitochondria with mostly s-Opa1 showed irregular cristae packing with wider cristae junctions and more narrow cristae than WT.BH3 profiling show Opa1-knock-out cells have reduced apoptotic priming and reduced sensitivity to apoptosis-inducing agents, and the presence l-Opa1 restores a WT protective apoptotic response.