bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒07‒03
forty-two papers selected by
Kelsey Fisher-Wellman, East Carolina University



  1. Methods Mol Biol. 2022 ;2497 1-10
      Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.
    Keywords:  Electron transport chain; Mitochondria; Mitochondrial membrane potential; Mitochondrial metabolism; NADH; Oxygen consumption; Respiratory complex; TMRM; Warburg Metabolism
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_1
  2. Front Oncol. 2022 ;12 816504
      Therapeutic targeting of tumor vulnerabilities is emerging as a key area of research. This review is focused on exploiting the vulnerabilities of tumor cells and the immune cells in the tumor immune microenvironment (TIME), including tumor hypoxia, tumor acidity, the bidirectional proton-coupled monocarboxylate transporters (MCTs) of lactate, mitochondrial oxidative phosphorylation (OXPHOS), and redox enzymes in the tricarboxylic acid cycle. Cancer cells use glucose for energy even under normoxic conditions. Although cancer cells predominantly rely on glycolysis, many have fully functional mitochondria, suggesting that mitochondria are a vulnerable target organelle in cancer cells. Thus, one key distinction between cancer and normal cell metabolism is metabolic reprogramming. Mitochondria-targeted small molecule inhibitors of OXPHOS inhibit tumor proliferation and growth. Another hallmark of cancer is extracellular acidification due lactate accumulation. Emerging results show that lactate acts as a fuel for mitochondrial metabolism and supports tumor proliferation and growth. Metabolic reprogramming occurs in glycolysis-deficient tumor phenotypes and in kinase-targeted, drug-resistant cancers overexpressing OXPHOS genes. Glycolytic cancer cells located away from the vasculature overexpress MCT4 transporter to prevent overacidification by exporting lactate, and the oxidative cancer cells located near the vasculature express MCT1 transporter to provide energy through incorporation of lactate into the tricarboxylic acid cycle. MCTs are, therefore, a vulnerable target in cancer metabolism. MCT inhibitors exert synthetic lethality in combination with metformin, a weak inhibitor of OXPHOS, in cancer cells. Simultaneously targeting multiple vulnerabilities within mitochondria shows synergistic antiproliferative and antitumor effects. Developing tumor-selective, small molecule inhibitors of OXPHOS with a high therapeutic index is critical to fully exploiting the mitochondrial vulnerabilities. We and others developed small-molecule inhibitors containing triphenylphosphonium cation that potently inhibit OXPHOS in tumor cells and tissues. Factors affecting tumor cell vulnerabilities also impact immune cells in the TIME. Glycolytic tumor cells supply lactate to the tumor-suppressing regulatory T cells overexpressing MCTs. Therapeutic opportunities for targeting vulnerabilities in tumor cells and the TIME, as well as the implications on cancer health disparities and cancer treatment, are addressed.
    Keywords:  Mitochondrial drugs; metabolic reprogramming; monocarboxylate transporters; oxidative phosphorylation (OXPHOS); tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2022.816504
  3. Front Oncol. 2022 ;12 919880
      Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.
    Keywords:  bioenergetics; cancer; hepatocellular carcinoma; metabolism; mitochondria
    DOI:  https://doi.org/10.3389/fonc.2022.919880
  4. Methods Mol Biol. 2022 ;2497 291-299
      The ubiquinone (Q) pool represents a node in the mitochondrial electron transport chain (ETC) onto which the electrons of all respiratory dehydrogenases converge. The redox state of the Q pool correlates closely with the electron flux through the ETC and is thus a parameter of great metabolic value for both the mitochondrial and cellular metabolism. Here, we describe the simultaneous measurement of respiratory rates of isolated mouse heart mitochondria and the redox state of their Q pool using a custom-made combination of a Clark-type oxygen electrode and a Q electrode.
    Keywords:  Mitochondria; Redox state; Respiratory rates; Ubiquinone pool
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_19
  5. Methods Mol Biol. 2022 ;2497 339-348
      Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.
    Keywords:  In-gel activity; Mitochondria; Oxidative phosphorylation; Primary fibroblasts; Supercomplex
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_25
  6. Methods Mol Biol. 2022 ;2497 363-422
      Mitochondria are complex organelles that use catabolic metabolism to produce ATP which is the critical energy source for cell function. Oxidative phosphorylation by the electron transport chain, which receives reducing equivalents (NADH and FADH2) from the tricarboxylic acid cycle, also produces reactive oxygen species (ROS) as a by-product at complex I and III. ROS play a significant role in health and disease. In order to better understand this process, a computational model of mitochondrial energy metabolism and the production of ROS has been developed. The model demonstrates the process regulating ROS production and removal and how different energy substrates can affect ROS production.
    Keywords:  Electron transport; Mitochondria; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_27
  7. Respir Physiol Neurobiol. 2022 Jun 28. pii: S1569-9048(22)00098-2. [Epub ahead of print] 103939
      While administration of the cyclic redox agent methylene blue (MB) during an intoxication by mitochondrial poisons (cyanide, hydrogen sulfide, rotenone) increases survival, the mechanisms behind these antidotal properties remain poorly understood. The objective of the studies presented in this paper was to characterize the interactions between the redox properties of MB, the intermediate metabolism and the mitochondrial respiration. We first show that intra-venous administration of micromolar levels of methylene blue in sedated and mechanically ventilated rats, increases not only resting oxygen consumption but also CO2 production (by ~ 50%), with no change in their ratio. This hypermetabolic state could be reproduced in a cellular model, where we found that the rate of electron transfer to MB was of the same order of magnitude as that of normal cellular metabolism. Notably, the large increase in cellular oxygen consumption caused by MB was relatively indifferent to the status of the mitochondrial respiratory chain: oxygen consumption persisted even when the respiratory chain was inhibited or absent (using inhibitors and cells deficient in mitochondrial oxidative phosphorylation); yet MB did not impede mitochondrial ATP production in control conditions. We present evidence that after being reduced into leuco-methylene blue (LMB) in presence of reducing molecules that are physiologically found in cells (such as NADH), the re-oxidation of LMB by oxygen can account for the increased oxygen consumption observed in vivo. In conditions of acute mitochondrial dysfunction, these MB redox cycling properties allow the rescue of the glycolysis activity and Krebs cycle through an alternate route of oxidation of NADH (or other potential reduced molecules), which accumulation would have otherwise exerted negative feedback on these metabolic pathways. Our most intriguing finding is that re-oxidization of MB by oxygen ultimately results in an in vivo matching between the increase in the rate of O2 consumed, by MB re-oxidation, and the rate of CO2, produced by the intermediate metabolism, imitating the fundamental coupling between the glycolysis/Krebs cycle and the mitochondrial respiration.
    Keywords:  Krebs cycle; Methylene blue; Mitochondrion
    DOI:  https://doi.org/10.1016/j.resp.2022.103939
  8. Nature. 2022 Jun 29.
      Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
    DOI:  https://doi.org/10.1038/s41586-022-04898-5
  9. Methods Mol Biol. 2022 ;2497 107-115
      The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.
    Keywords:  Assembly; Blue Native Gel; Brain; Muscle; Respiratory complex
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_7
  10. J Cell Sci. 2022 Jul 01. pii: jcs.259090. [Epub ahead of print]
      Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the therapeutic strategies targeting this vulnerability are still inefficient and have unacceptable side effects in clinical trials. Therefore, developing biomarkers to predict therapeutic efficacy would be essential to improve the selective targeting of cancer cells. Here, we found that the cell lines sensitive to glucose deprivation have high expression of cystine/glutamate antiporter xCT. We found that cystine uptake and glutamate export through xCT contributed to rapid NADPH depletion under glucose deprivation. This collapse of the redox system oxidized and inactivated AMPK, a major regulator of metabolic adaptation, resulting in a metabolic catastrophe and cell death. While this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. Taken together, these findings suggest a novel cross-talk between AMPK and xCT for the metabolism and signal transduction and reveal a metabolic vulnerability in xCT-high expressing cancer cells to glucose deprivation.
    Keywords:  AMPK; Cystine; Glucose starvation; NADPH; SLC7A11; xCT
    DOI:  https://doi.org/10.1242/jcs.259090
  11. J Biol Chem. 2022 Jun 24. pii: S0021-9258(22)00638-X. [Epub ahead of print] 102196
      In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared to the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.
    Keywords:  2′-3′-dideoxycytidine (ddC, zalcitabine); Mitochondrial DNA (mtDNA) maintenance; POLG c.2864A>G/p.Y955C; SJCRH30; autosomal dominant progressive external ophthalmoplegia (adPEO); cell line model of mitochondrial disease; mitochondrial toxicity
    DOI:  https://doi.org/10.1016/j.jbc.2022.102196
  12. Methods Mol Biol. 2022 ;2497 185-206
      Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).
    Keywords:  Bioenergetics; Cellular respiration; Mitochondrial respiration; Permeabilized cells; Seahorse XF Extracellular Flux Analyzer
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_12
  13. Methods Mol Biol. 2022 ;2497 325-332
      Mitochondrial Ca2+ buffering is a hallmark of eukaryotic cellular physiology, contributing to the spatiotemporal shaping of the cytosolic Ca2+ signals and regulation of mitochondrial bioenergetics. Often, this process is altered in a pathological context; therefore, it can be scrutinized experimentally for therapeutic intervention. In this chapter, we describe fluorescence and bioluminescence measurement of mitochondrial Ca2+ in both isolated mitochondria and intact cells.
    Keywords:  Bioluminescence calcium sensing; Calcium-sensitive genetic probes; Fluorescence calcium imaging; Mitochondrial calcium
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_23
  14. Methods Mol Biol. 2022 ;2497 141-172
      Mitochondrial energy production is crucial for normal daily activities and maintenance of life. Herein, the logic and execution of two main classes of measurements are outlined to delineate mitochondrial function: ATP production and oxygen consumption. Aerobic ATP production is quantified by phosphorus magnetic resonance spectroscopy (31PMRS) in vivo in both human subjects and animal models using the same protocols and maintaining the same primary assumptions. Mitochondrial oxygen consumption is quantified by oxygen polarography and applied in isolated mitochondria, cultured cells, and permeabilized fibers derived from human or animal tissue biopsies. Traditionally, mitochondrial functional measures focus on maximal oxidative capacity-a flux rate that is rarely, if ever, observed outside of experimental conditions. Perhaps more physiologically relevant, both measurement classes herein focus on one principal design paradigm; submaximal mitochondrial fluxes generated by graded levels of ADP to map the function for ADP sensitivity. We propose this function defines the bioenergetic role that mitochondria fill within the myoplasm to sense and match ATP demands. Any deficit in this vital role for ATP homeostasis leads to symptoms often seen in cardiovascular and cardiopulmonary diseases, diabetes, and metabolic syndrome.
    Keywords:  ADP sensitivity; Aerobic metabolism; Bioenergetics; Free energy homeostasis; Magnetic resonance; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_10
  15. Commun Biol. 2022 Jul 01. 5(1): 649
      Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.
    DOI:  https://doi.org/10.1038/s42003-022-03606-3
  16. Methods Mol Biol. 2022 ;2497 313-318
      This section aims to describe the measurement of NADH and FAD2+ levels in intact cells using fluorescence microscopy. Both NADH and FADH2 are major electron donors for the electron transport chain through shifting of their redox status. Furthermore, within their redox couples, only NADH and FAD2+ are fluorescent. Therefore, calibration of the NADH and FAD2+ fluorescence signal is a crucial factor in accurately assessing mitochondrial function and redox status.
    Keywords:  Autofluorescence; ETC; FAD2+; NADH; Redox status
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_21
  17. J Am Heart Assoc. 2022 Jun 29. e026135
      Background The metabolite succinate accumulates during cardiac ischemia. Within 5 minutes of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II. The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking MCT1 (monocarboxylate transporter 1) may be beneficial in IR injury, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT1 would retain succinate in cells, exacerbating ROS generation and IR injury. Methods and Results Using the mitochondrial ROS probe mitoSOX and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in situ during the first moments of reperfusion. We found that acute MCT1 inhibition enhanced mitochondrial ROS generation at reperfusion and worsened IR injury (recovery of function and infarct size). Both of these effects were abrogated by tandem inhibition of mitochondrial complex II, suggesting that succinate retention worsens IR because it drives more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator tetramethylrhodamine ethyl ester, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT1 inhibition. Conclusions Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation as key determinants of PT pore opening and IR injury outcomes.
    Keywords:  complex II; ischemia; metabolism; mitochondria; reactive oxygen species; succinate
    DOI:  https://doi.org/10.1161/JAHA.122.026135
  18. Mol Cell. 2022 Jun 18. pii: S1097-2765(22)00541-X. [Epub ahead of print]
      Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.
    Keywords:  PKM2; cancer metabolism; glucose oxidation; metastasis; methionine oxidation; pancreatic cancer; redox signaling
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.005
  19. Proc Natl Acad Sci U S A. 2022 Jul 05. 119(27): e2123090119
      Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
    Keywords:  NADH dehydrogenase; biological energy conversion; complex I; proton translocation; respiratory chain
    DOI:  https://doi.org/10.1073/pnas.2123090119
  20. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]
      Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
    Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.004
  21. Angew Chem Int Ed Engl. 2022 Jun 27.
      The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is known to be integral to a wide variety of biological cellular activities, yet to date there are no analytical methods availab le to directly detect the ratio of intracellular GTP to GDP. Herein, we report GRISerHR, a genetically encoded fluorescent biosensor to monitor the GTP:GDP ratio  in multiple cell types and in various organelles under metabolic perturbation. Additionally, we characterized the differential mitochondrial GTP:GDP ratios resulting from genetic modulation of two isoforms of a tricarboxylic acid (TCA) cycle enzyme (succinyl-CoA synthetase; SCS-ATP and SCS-GTP) and of a phosphoenolpyruvate (PEP) cycle enzyme (PEPCK-M). Thus, our GRISerHR sensor achieves spatiotemporally precise detection of dynamic changes in the endogenous GTP:GDP ratio in living cells and can help deepen our understanding about the energy metabolic contributions of guanosine nucleotides in biology.
    Keywords:  GTP:GDP ratio biosensor imaging mitochondria metabolism
    DOI:  https://doi.org/10.1002/anie.202201266
  22. Methods Mol Biol. 2022 ;2497 11-61
      The mitochondrial membrane potential (ΔψM) is the major component of the bioenergetic driving force responsible for most cellular ATP produced, and it controls a host of biological processes. In intact cells, assay readouts with commonly used fluorescence ΔψM probes are distorted by factors other than ΔψM. Here, we describe a protocol to calculate both ΔψM and plasma membrane potential (ΔψP) in absolute millivolts in intact single cells, or in populations of adherent, cultured cells. Our approach generates unbiased data that allows comparison of ΔψM between cell types with different geometry and ΔψP, and to follow ΔψM in time when ΔψP fluctuates. The experimental paradigm results in fluorescence microscopy time courses using a pair of cationic and anionic probes with internal calibration points that are subsequently computationally converted to millivolts on an absolute scale. The assay is compatible with wide field, confocal or two-photon microscopy. The method given here is optimized for a multiplexed, partial 96-well microplate format to record ΔψP and ΔψM responses for three consecutive treatment additions.
    Keywords:  Bis-oxonol; Cell culture; Cellular heterogeneity; Fluorescence microscopy; Live cell microscopy; MitoTracker; Mitochondrial biogenesis; Mitochondrial membrane potential; Plasma membrane potential; Single cell; TMRM; Tetramethylrhodamine methyl ester
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_2
  23. Nat Commun. 2022 Jun 28. 13(1): 3702
      The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.
    DOI:  https://doi.org/10.1038/s41467-022-31462-6
  24. Methods Mol Biol. 2022 ;2497 319-324
      The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III, and IV) is an essential component in the process of energy generation during oxidative phosphorylation. Tetramethylrhodamine, methyl ester, perchlorate (TMRM) is one of the most commonly used fluorescent reporters of ΔΨm. TMRM is routinely employed in a steady state for the measurement of membrane potential. However, it can also be utilized with time-lapse fluorescence imaging to effectively monitor the changes in membrane potential in response to a given stimulus by analyzing the change in distribution of the dye with time.
    Keywords:  Fluorescence microscopy; Mitochondria membrane potential; Primary skin fibroblasts; TMRM; Uncoupler
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_22
  25. Endocrinology. 2022 Jun 26. pii: bqac094. [Epub ahead of print]
      Immune cells infiltrate adipose tissue as a function of age, sex, and diet leading to a variety of regulatory processes linked to metabolic disease and dysfunction. Cytokines and chemokines produced by resident macrophages, B cells, T cells and eosinophils play major role(s) in fat cell mitochondrial functions modulating pyruvate oxidation, electron transport and oxidative stress, branched chain amino acid (BCAA) metabolism, fatty acid oxidation and apoptosis. Indeed, cytokine-dependent down regulation of numerous genes affecting mitochondrial metabolism is strongly linked to the development of the metabolic syndrome while in contrast, the potentiation of mitochondrial metabolism represents a counter regulatory process improving metabolic outcomes. In contrast, inflammatory cytokines activate mitochondrially-linked cell death pathways such as apoptosis, pyroptosis, necroptosis and ferroptosis. As such, the adipocyte mitochondrion represents a major intersection point for immunometabolic regulation of central metabolism.
    Keywords:  Adipose; Inflammation; Macrophage; Mitochondria
    DOI:  https://doi.org/10.1210/endocr/bqac094
  26. Nat Metab. 2022 Jun;4(6): 739-758
      Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.
    DOI:  https://doi.org/10.1038/s42255-022-00593-x
  27. J Mater Chem B. 2022 Jul 01.
      Cellular dysregulated pH and mitochondrial metabolism are commonly two central factors for solid tumour progression. pH regulation and long-term mitochondrial tracking provide a great opportunity for tumour treatment. pH probes with suitable pKa and satisfactory mitochondria-immobilizing character are demanded for tumour recognition and therapy. Here, we report a ratiometric fluorescent probe, CouDa, for pH imaging in mitochondria and tumour tissue. CouDa displays an obvious blue-shifted emission (about 160 nm) in alkaline environment, with a pKa around 7.4 suitable for monitoring mitochondrial pH change. NMR and MS data confirmed an addition reaction mechanism of OH- upon the positively charged conjugated structure of hemicyanine fluorophore. Mitochondrial immobilization and acidification monitoring were realized by CouDa in cells treated with a mitochondrial uncoupler. Moreover, CouDa could distinguish acidified tumour tissue in living mice. Comparing with its analogue, the pH-sensitivity and mitochondria-immobilizing property are attributed to a hydrophobic long alkyl chain on indolium N atom. This work provides an effective strategy to track nucleophilic substances in dysfunctional mitochondria.
    DOI:  https://doi.org/10.1039/d2tb00553k
  28. J Clin Invest. 2022 Jul 01. pii: e158447. [Epub ahead of print]132(13):
      Mitochondrial dysfunction and cell senescence are hallmarks of aging and are closely interconnected. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity per mitochondrion together with a decreased mitochondrial membrane potential, typically accompanied by increased production of oxygen free radicals, is a cause and a consequence of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. Here, we summarize pathways that cause mitochondrial dysfunction in senescence and aging and discuss the major consequences of mitochondrial dysfunction and how these consequences contribute to senescence and aging. We also highlight the potential of senescence-associated mitochondrial dysfunction as an antiaging and antisenescence intervention target, proposing the combination of multiple interventions converging onto mitochondrial dysfunction as novel, potent senolytics.
    DOI:  https://doi.org/10.1172/JCI158447
  29. Nat Commun. 2022 Jun 27. 13(1): 3682
      The bacterial respiratory electron transport system (ETS) is branched to allow condition-specific modulation of energy metabolism. There is a detailed understanding of the structural and biochemical features of respiratory enzymes; however, a holistic examination of the system and its plasticity is lacking. Here we generate four strains of Escherichia coli harboring unbranched ETS that pump 1, 2, 3, or 4 proton(s) per electron and characterized them using a combination of synergistic methods (adaptive laboratory evolution, multi-omic analyses, and computation of proteome allocation). We report that: (a) all four ETS variants evolve to a similar optimized growth rate, and (b) the laboratory evolutions generate specific rewiring of major energy-generating pathways, coupled to the ETS, to optimize ATP production capability. We thus define an Aero-Type System (ATS), which is a generalization of the aerobic bioenergetics and is a metabolic systems biology description of respiration and its inherent plasticity.
    DOI:  https://doi.org/10.1038/s41467-022-30877-5
  30. Cell Rep. 2022 Jun 28. pii: S2211-1247(22)00801-4. [Epub ahead of print]39(13): 111012
      Ovarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of OC-related deaths. In particular, OC tumors preferentially metastasize to and proliferate rapidly in the omentum. Here, we show that metastatic OC cells experience increased oxidative stress in the omental microenvironment. Metabolic reprogramming, including upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, allows OC cells to compensate for this challenge. Inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting that this adaptive metabolic dependency is important for OC omental metastasis.
    Keywords:  CP: Cancer; CP: Metabolism; metabolism; metastasis; ovarian cancer
    DOI:  https://doi.org/10.1016/j.celrep.2022.111012
  31. Cancer Discov. 2022 Jun 30. pii: candisc.0043.2022-1-10 21:36:20.420. [Epub ahead of print]
      Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC we now demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in PDAC patients. Together, our data reveal that maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC.
    DOI:  https://doi.org/10.1158/2159-8290.CD-22-0043
  32. Sci Rep. 2022 Jun 27. 12(1): 10877
      The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.
    DOI:  https://doi.org/10.1038/s41598-022-14860-0
  33. Cancer Med. 2022 Jun 27.
      BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer.METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer.
    RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown.
    CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.
    Keywords:  E2F1; PCK2; estrogen receptor positive (ER+) breast cancer; mTORC1; mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M)
    DOI:  https://doi.org/10.1002/cam4.4969
  34. Trends Cell Biol. 2022 Jun 23. pii: S0962-8924(22)00138-6. [Epub ahead of print]
      While once regarded as ATP factories, mitochondria have taken the spotlight as important regulators of cellular homeostasis. The past two decades have witnessed an intensifying interest in the study of mitochondria in cells of the immune system, with many new and unexpected roles for mitochondria emerging. Immune cells offer intriguing insights as mitochondria appear to play different roles at different stages of T cell development, matching the changing functions of the cells. Here we briefly review the multifaceted roles of mitochondria during T cell differentiation, focusing on CD8+ cytotoxic T lymphocytes (CTLs) and we consider how mitochondrial dysfunction can contribute to CTL exhaustion. In addition, we highlight a newly appreciated role for mitochondria as homeostatic regulators of CTL-mediated killing and explore the emerging literature describing mechanisms linking cytosolic and mitochondrial protein synthesis.
    Keywords:  CD8; CTL; T cell; exhaustion; mitochondria; mitochondrial translation
    DOI:  https://doi.org/10.1016/j.tcb.2022.05.007
  35. Free Radic Res. 2022 Jun 30. 1-15
      To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP+) as a mitochondrial vector forming PB-TPP+ probes. Two pyrene isomers with the n-butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP+-based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP+ vector, a warning to the user of this well-known vector.
    Keywords:  Fluorescent probe; pyrene; reactive oxygen species; time-resolved spectroscopy; triphenylphosphonium salt
    DOI:  https://doi.org/10.1080/10715762.2022.2077202
  36. J Biol Chem. 2022 Jun 25. pii: S0021-9258(22)00635-4. [Epub ahead of print] 102193
      Macrophages respond to their environment by adopting a predominantly inflammatory or anti-inflammatory profile, depending on the context. The polarization of the subsequent response is regulated by a combination of intrinsic and extrinsic signals and is associated with alterations in macrophage metabolism. Although macrophages are important producers of Wnt ligands, the role of Wnt signaling in regulating metabolic changes associated with macrophage polarization remains unclear. Wnt4 upregulation has been shown to be associated with tissue repair and suppression of age-associated inflammation, which led us to generate Wnt4-deficient bone marrow-derived macrophages (BMDMs) to investigate its role in metabolism. We show that loss of Wnt4 led to modified mitochondrial structure, enhanced oxidative phosphorylation, and depleted intracellular lipid reserves, as the cells depended on fatty acid oxidation to fuel their mitochondria. Further we found that enhanced lipolysis was dependent on protein kinase C (PKC)-mediated activation of lysosomal acid lipase in Wnt4-deficient BMDMs. Although not irreversible, these metabolic changes promoted parasite survival during infection with Leishmania donovani. In conclusion, our results indicate that enhanced macrophage fatty acid oxidation impairs the control of intracellular pathogens, such as Leishmania. We further suggest that Wnt4 may represent a potential target in atherosclerosis, which is characterized by lipid storage in macrophages leading to them becoming foam cells.
    Keywords:  BMDM; Leishmania; Wnt4 signaling; fatty acid oxidation; lipid droplet; macrophage
    DOI:  https://doi.org/10.1016/j.jbc.2022.102193
  37. Methods Mol Biol. 2022 ;2497 117-128
      The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O2 consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.
    Keywords:  Brain; Electron transport chain; Muscle; O2 consumption; Seahorse
    DOI:  https://doi.org/10.1007/978-1-0716-2309-1_8
  38. Cell Death Dis. 2022 Jun 30. 13(6): 576
      The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.
    DOI:  https://doi.org/10.1038/s41419-022-05028-9
  39. Nat Cancer. 2022 Jun 30.
      Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.
    DOI:  https://doi.org/10.1038/s43018-022-00402-0
  40. Mol Med Rep. 2022 Aug;pii: 268. [Epub ahead of print]26(2):
      Under aerobic conditions, the preferential use of anaerobic glycolysis by tumour cells leads to a high level of lactate accumulation in tumour microenvironment. Lactate acts not only as a cellular energy source but also as a signalling molecule that regulates cancer cell growth, metastasis and metabolism. It has been reported that a G‑protein‑coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) is highly expressed in numerous types of cancer, but the detailed mechanism remains unclear. In the present study, it was reported that HCAR1 is highly expressed in breast cancer cells. Genetic deletion of HCAR1 in MCF7 cells leads to reduced cell proliferation and migration. Moreover, it was observed that knockout (KO) of HCAR1 attenuated the expression and activity of phosphofructokinase and hexokinase, key rate‑limiting enzymes in glycolysis. Using an extracellular flux analyzer, it was showed that KO of HCAR1 promoted a metabolic shift towards a decreased glycolysis state, as evidenced by a decreased extracellular acidification rate and increased oxygen consumption rate in MCF7 cells. Taken together, our results suggested that lactate acts through HCAR1 as a metabolic regulator in breast cancer cells that may be therapeutically exploited.
    Keywords:  HCAR1; breast cancer; energy metabolism; metastases
    DOI:  https://doi.org/10.3892/mmr.2022.12784
  41. Nat Metab. 2022 Jun;4(6): 693-710
      Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.
    DOI:  https://doi.org/10.1038/s42255-022-00582-0