bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2021‒06‒13
forty-six papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Sci Signal. 2021 Jun 08. pii: eabc7405. [Epub ahead of print]14(686):
      Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD+ concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.
  2. Annu Rev Cancer Biol. 2021 Mar;5(1): 235-257
      Metabolic and epigenetic reprogramming are characteristics of cancer cells that, in many cases, are linked. Oncogenic signaling, diet, and tumor microenvironment each influence the availability of metabolites that are substrates or inhibitors of epigenetic enzymes. Reciprocally, altered expression or activity of chromatin-modifying enzymes can exert direct and indirect effects on cellular metabolism. In this article, we discuss the bidirectional relationship between epigenetics and metabolism in cancer. First, we focus on epigenetic control of metabolism, highlighting evidence that alterations in histone modifications, chromatin remodeling, or the enhancer landscape can drive metabolic features that support growth and proliferation. We then discuss metabolic regulation of chromatin-modifying enzymes and roles in tumor growth and progression. Throughout, we highlight proposed therapeutic and dietary interventions that leverage metabolic-epigenetic cross talk and have the potential to improve cancer therapy.
    Keywords:  cancer; cell metabolism; chromatin modification; epigenetics
  3. Methods Mol Biol. 2021 ;2310 113-159
      Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.
    Keywords:  Cardiovascular diseases (CVD); Homeostasis; Metabolism; Mitochondrial morphology; Mitochondrial quality control; Pathology
  4. Adv Protein Chem Struct Biol. 2021 ;pii: S1876-1623(21)00025-0. [Epub ahead of print]126 307-343
      The maintenance of cellular homeostasis involves the participation of multiple organelles, such as the endoplasmic reticulum (ER) and mitochondria. Specifically, ER plays a key role in calcium (Ca2+) storage, lipid synthesis, protein folding, and assembly, while mitochondria are the "energy factories" and provide energy to drive intracellular processes. Hence, alteration in ER or mitochondrial homeostasis has detrimental effects on cell survival, being linked to the triggering of apoptosis, a programmed form of cell death. Besides, ER stress conditions affect mitochondria functionality and vice-versa, as ER and mitochondria communicate via mitochondria-associated ER membranes (MAMs) to carry out a number of fundamental cellular functions. It is not surprising, thus, that also MAMs perturbations are involved in the regulation of apoptosis. This chapter intends to accurately discuss the involvement of MAMs in apoptosis, highlighting their crucial role in controlling this delicate cellular process.
    Keywords:  Apoptosis; Bioenergetics; Calcium signaling; ER-mitochondria contact sites; MAMs; Organelle tethering
  5. Methods Mol Biol. 2021 ;2275 363-378
      In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.
    Keywords:  ER–Mitochondria tethering; Endoplasmic reticulum; Mitochondria; Organelle contact sites; SPLICS; Split GFP
  6. Front Oncol. 2021 ;11 639408
      Metabolic reprogramming is the prominent feature of clear cell renal cell carcinoma (ccRCC). Succinate dehydrogenase subunit B (SDHB) is one of subunits of mitochondrial respiratory chain complex II. The loss of SDHB function is closely related with metabolic changes in kidney cancer cells. However, the role and molecular mechanism of SDHB in ccRCC occurrence and progression are still unclear. In this study, the results of bioinformatics analyses on GEO, TCGA and oncomine databases and immunohistochemistry showed that the expression level of SDHB was downregulated in ccRCC tissues. SDHB level was gradually downregulated as ccRCC stage and grade progressed. The low level of SDHB was associated with poor prognosis of ccRCC patients, especially for advanced ccRCC patients. Increased methylation levels in SDHB gene promoter led to the downregulation of SDHB level in ccRCC tissues. SDHB was correlated with many metabolism related genes and its interacting proteins were enriched in metabolic pathways. SDHB overexpression suppressed the proliferation, colony formation and migration of ccRCC cells by inhibiting aerobic glycolysis. SDHB may be a potential prognostic marker and therapeutic target for ccRCC.
    Keywords:  SDHB; glycolysis; methylation; prognosis; renal cell carcinoma
  7. Immunity. 2021 May 28. pii: S1074-7613(21)00209-0. [Epub ahead of print]
      A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.
    Keywords:  CD36; CD8(+) T cells; lipid peroxidation; oxidized lipids; tumor microenvironment
  8. Cell Rep. 2021 Jun 08. pii: S2211-1247(21)00562-3. [Epub ahead of print]35(10): 109212
      Obesity is an established risk factor for cancer in many tissues. In the mammalian intestine, a pro-obesity high-fat diet (HFD) promotes regeneration and tumorigenesis by enhancing intestinal stem cell (ISC) numbers, proliferation, and function. Although PPAR (peroxisome proliferator-activated receptor) nuclear receptor activity has been proposed to facilitate these effects, their exact role is unclear. Here we find that, in loss-of-function in vivo models, PPARα and PPARδ contribute to the HFD response in ISCs. Mechanistically, both PPARs do so by robustly inducing a downstream fatty acid oxidation (FAO) metabolic program. Pharmacologic and genetic disruption of CPT1A (the rate-controlling enzyme of mitochondrial FAO) blunts the HFD phenotype in ISCs. Furthermore, inhibition of CPT1A dampens the pro-tumorigenic consequences of a HFD on early tumor incidence and progression. These findings demonstrate that inhibition of a HFD-activated FAO program creates a therapeutic opportunity to counter the effects of a HFD on ISCs and intestinal tumorigenesis.
    Keywords:  Apc; Cpt1a; Ppar; fatty acid oxidation; high-fat diet; intestinal stem cells
  9. Methods Mol Biol. 2021 ;2310 271-285
      NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.
    Keywords:  18O-NAD+; Diaphorase; HPLC; Isotopes; LC-MS; Lactate; Lactate dehydrogenase (LDH); Mitochondria isolation; NAD+; NAD+/NADH cycling assay; Resazurin; Resorufin
  10. Development. 2021 Jun 01. pii: dev199609. [Epub ahead of print]148(11):
      The intimate relationships between cell fate and metabolism have long been recognized, but a mechanistic understanding of how metabolic pathways are dynamically regulated during development and disease, how they interact with signalling pathways, and how they affect differential gene expression is only emerging now. We summarize the key findings and the major themes that emerged from the virtual Keystone Symposium 'Metabolic Decisions in Development and Disease' held in March 2021.
    Keywords:  Cell fate; Development; Metabolic plasticity; Metabolism; Nutrition
  11. Semin Cancer Biol. 2021 Jun 04. pii: S1044-579X(21)00172-3. [Epub ahead of print]
      Recurrent disease after prolonged tumor dormancy is a major cause of cancer associated mortality, yet many of the mechanisms that are engaged to initiate dormancy as well as later recurrence remain incompletely understood. It is known that cancer cells initiate adaptation mechanisms to adapt tightly regulated cellular processes to non-optimal growth environments; Recent investigations have begun to elucidate the contribution of these mechanisms to malignant progression, with intriguing studies now defining cellular stress as a key contributor to the development and maintenance of cancer dormancy. This review will discuss our current understanding of stress responses facilitating malignant cell adaptation and metabolic reprogramming to establish tumor dormancy.
    Keywords:  Cancer dormancy; Cancer recurrence; Endoplasmic Reticulum stress; Metabolism; Oxidative Stress
  12. Aging Cell. 2021 Jun 07. e13408
      Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep / ep ) or hyper-accurate (Mrps12ha / ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep / ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha / ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging.
    Keywords:  ageing; metabolism; mitochondria; protein synthesis
  13. Methods Mol Biol. 2021 ;2310 259-270
      Mitochondria play a central role in metabolic reprograming that occurs in numerous disease conditions. A precise evaluation of the extent of mitochondrial involvement in the metabolic alterations is essential for a better definition of metabolically based therapeutic strategies. In this chapter, some simple protocols are presented, using carbon 13 tracers and nuclear magnetic resonance isotopomer analysis, for the evaluation of mitochondrial contributions to intermediary metabolism and the metabolic effects of the implementation of some mitochondrial regulatory mechanisms.
    Keywords:  13C isotope tracers; Intermediary metabolism; Metabolic reprograming; Mitochondria; Mitochondrial regulation; NMR isotopomer analysis; TCA cycle
  14. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2020078118. [Epub ahead of print]118(24):
      Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.
    Keywords:  antipsychotic drugs; autophagy; mitochondria; multiple sclerosis; remyelination
  15. Redox Biol. 2021 Jun 01. pii: S2213-2317(21)00179-8. [Epub ahead of print]45 102021
      Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.
    Keywords:  Ferroptosis; Glutathione; Heart; Ischemia-reperfusion; Mitochondria; Oxidized phosphatidylethanolamine
  16. Front Physiol. 2021 ;12 675708
      Mitochondrial porins, also known as voltage-dependent anion selective channels (VDACs), are pore-forming molecules of the outer mitochondrial membranes, involved in the regulation of metabolic flux between cytosol and mitochondria. Playing such an essential role, VDAC proteins are evolutionary conserved and isoforms are present in numerous species. The quest for specific function(s) related to the raise of multiple isoforms is an intriguing theme. The yeast Saccharomyces cerevisiae genome is endowed with two different VDAC genes encoding for two distinct porin isoforms, definitely less characterized in comparison to mammalian counterpart. While yVDAC1 has been extensively studied, the second isoform, yVDAC2, is much less expressed, and has a still misunderstood function. This review will recapitulate the known and poorly known information in the literature, in the light of the growing interest about the features of VDAC isoforms in the cell.
    Keywords:  VDAC; electrophyiology; mitochondria; outer mitochondrial membrane; porin; yeast
  17. Immunity. 2021 Jun 10. pii: S1074-7613(21)00210-7. [Epub ahead of print]
      Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.
    Keywords:  ARDS; CMPK2; COVID-19; IL-1β; IL-6; NLRP3 inflammasome; SARS-CoV-2; inflammation; metformin; mitochondrial DNA
  18. J Cell Sci. 2021 Jun 01. pii: jcs253781. [Epub ahead of print]134(11):
      In Saccharomyces cerevisiae, the selective autophagic degradation of mitochondria, termed mitophagy, is critically regulated by the adapter protein Atg32. Despite our knowledge about the molecular mechanisms by which Atg32 controls mitophagy, its physiological roles in yeast survival and fitness remains less clear. Here, we demonstrate a requirement for Atg32 in promoting spermidine production during respiratory growth and heat-induced mitochondrial stress. During respiratory growth, mitophagy-deficient yeast exhibit profound heat-stress induced defects in growth and viability due to impaired biosynthesis of spermidine and its biosynthetic precursor S-adenosyl methionine. Moreover, spermidine production is crucial for the induction of cytoprotective nitric oxide (NO) during heat stress. Hence, the re-addition of spermidine to Atg32 mutant yeast is sufficient to both enhance NO production and restore respiratory growth during heat stress. Our findings uncover a previously unrecognized physiological role for yeast mitophagy in spermidine metabolism and illuminate new interconnections between mitophagy, polyamine biosynthesis and NO signaling.
    Keywords:  ATG32; Autophagy; Mitophagy; Nitric oxide; S-adenosyl methionine; Spermidine
  19. Methods Mol Biol. 2021 ;2310 247-258
      We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.
    Keywords:  Brown adipose tissue; Complex 1; Complex 2; Mitochondria; NDUFA9; Reactive oxygen species; Succinate; UCP1
  20. iScience. 2021 May 21. 24(5): 102494
      Dihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance.
    Keywords:  Biological sciences; Cellular physiology; Diabetology; Endocrinology; Physiology
  21. Dev Cell. 2021 Jun 01. pii: S1534-5807(21)00439-1. [Epub ahead of print]
      Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.
    Keywords:  AMPK; PI(5)P; PIKfyve; ULK1; autophagy; glucose starvation; mTOR; phagophore
  22. Methods Mol Biol. 2021 ;2310 301-309
      Metabolic flexibility is vital for organisms to respond to and survive changes in energy availability. A critical metabolic flexibility regulator is peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which regulates various transcription factors and nuclear receptors that, in turn, regulate mitochondrial homeostasis and fatty acid oxidation. PGC-1α is itself regulated, with one of the significant modes of regulation being acetylation. Thus, measuring the acetylation status of PGC-1α is a critical indicator of cells' metabolic flexibility. In this chapter, we describe a method of evaluating PGC-1α acetylation in primary mouse myotubes. This method can also be used with other cell types and tissues.
    Keywords:  Acetylation; Aging; Cachexia; Diabetes; GCN5; Mitochondrial biogenesis; PGC-1α; Respiration; SIRT1; Sarcopenia; Sirtuin deacetylase
  23. Nat Commun. 2021 06 08. 12(1): 3428
      Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.
  24. Methods Mol Biol. 2021 ;2310 287-299
      The dynamism of mitochondria, considered as complex and motile organelles, is brought about by mitochondria ability to undergo cycles of fission and fusion events, whose fine balance determines their morphology in a specific physiological context. A huge body of evidence makes it possible to associate mitochondrial organization to regulation of an increasing number of key cellular processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, mtDNA homeostasis, autophagy, and cell death. Here, we review the recently developed imaging methods for studying mitochondrial dynamics, including live-cell imaging, by using mitochondrial-targeted fluorescent proteins. In more details, we focus our attention on two different protocols in the T cell model, an example of nonadherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. Also, we discuss some examples of mouse models carrying mitochondria-targeted fluorescent proteins, which allow to investigate the mitochondrial morphology in vivo.
    Keywords:  Dynamic network; Fission; Fusion; Imaging; Mitochondria; Morphology
  25. EMBO Rep. 2021 Jun 08. e52032
      The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non-cell-autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF-κB signaling upregulation is central to elicit this immune response. Inactivating NF-κB abolishes NK cell-mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF-κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell-mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF-κB-mediated immunogenicity.
    Keywords:  NF-κB; aneuploidy; complex karyotypes; immune clearance; senescence
  26. Dev Cell. 2021 Jun 03. pii: S1534-5807(21)00440-8. [Epub ahead of print]
      Cells in tissues experience a plethora of forces that regulate their fate and modulate development and homeostasis. Cells sense mechanical cues through localized mechanoreceptors or by influencing cytoskeletal or plasma membrane organization. Cells translate force and modulate their behavior through a process termed mechanotransduction. Cells tune their tension upon exposure to chronic force by engaging cellular machinery that modulates actin tension, which in turn stimulates matrix remodeling and stiffening and alters cell-cell adhesions until cells achieve a state of tensional homeostasis. Loss of tensional homeostasis can be induced through oncogene activity and/or tissue fibrosis, accompanies tumor progression, and is associated with increased cancer risk. The mechanical stresses that develop in tumors can also foster the mesenchymal-like transdifferentiation of cells to induce a stem-like phenotype that contributes to their aggression, metastatic dissemination, and treatment resistance. Thus, strategies that ameliorate tumor mechanics may comprise an effective strategy to prevent aggressive tumor behavior.
  27. Sci Rep. 2021 Jun 11. 11(1): 12387
      Metabolic and bioenergetic plasticity of immune cells is essential for optimal responses to bacterial infections. AMPK and Parkin ubiquitin ligase are known to regulate mitochondrial quality control mitophagy that prevents unwanted inflammatory responses. However, it is not known if this evolutionarily conserved mechanism has been coopted by the host immune defense to eradicate bacterial pathogens and influence post-sepsis immunosuppression. Parkin, AMPK levels, and the effects of AMPK activators were investigated in human leukocytes from sepsis survivors as well as wild type and Park2-/- murine macrophages. In vivo, the impact of AMPK and Parkin was determined in mice subjected to polymicrobial intra-abdominal sepsis and secondary lung bacterial infections. Mice were treated with metformin during established immunosuppression. We showed that bacteria and mitochondria share mechanisms of autophagic killing/clearance triggered by sentinel events that involve depolarization of mitochondria and recruitment of Parkin in macrophages. Parkin-deficient mice/macrophages fail to form phagolysosomes and kill bacteria. This impairment of host defense is seen in the context of sepsis-induced immunosuppression with decreased levels of Parkin. AMPK activators, including metformin, stimulate Parkin-independent autophagy and bacterial killing in leukocytes from post-shock patients and in lungs of sepsis-immunosuppressed mice. Our results support a dual role of Parkin and AMPK in the clearance of dysfunctional mitochondria and killing of pathogenic bacteria, and explain the immunosuppressive phenotype associated Parkin and AMPK deficiency. AMPK activation appeared to be a crucial therapeutic target for the macrophage immunosuppressive phenotype and to reduce severity of secondary bacterial lung infections and respiratory failure.
  28. Cancer Res. 2021 Jun 07. pii: canres.2114.2020. [Epub ahead of print]
      Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of BRAFV600E cutaneous metastatic melanoma (CMM) patients eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming which profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafemib-sensitive and -resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers.
  29. Nat Commun. 2021 06 07. 12(1): 3333
      Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.
  30. Adv Sci (Weinh). 2021 06;8(11): e2003732
      Extracellular glutamine represents an important energy source for many cancer cells and its metabolism is intimately involved in maintaining redox homeostasis. The heightened metabolic activity within tumor tissues can result in glutamine deficiency, necessitating metabolic reprogramming responses. Here, dual mechanisms involving the stress-responsive transcription factor DDIT3 (DNA damage induced transcript 3) that establishes an interrelationship between glycolysis and mitochondrial respiration are revealed. DDIT3 is induced during glutamine deprivation to promote glycolysis and adenosine triphosphate production via suppression of the negative glycolytic regulator TIGAR. In concert, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1-mediated down-regulation of COQ9 and COX4. This in turn dampens the sustained levels of reactive oxygen species that follow glutamine withdrawal. Together these mechanisms constitute an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation.
    Keywords:  COQ9; COX4; DDIT3/CHOP; electron transfer chain; glutamine deprivation; glycolysis
  31. Am J Physiol Cell Physiol. 2021 06 09.
      Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, MELAS patients treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in MELAS patients are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared to control, healthy individuals. We observed that mitochondria from patients were fragmented, had a 3-fold decline in the average speed of motility, a 2-fold reduced mitochondrial membrane potential and a 1.5-2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared to healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 hrs resulted in increased mitochondrial respiration compared to control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.
    Keywords:  human fibroblasts; lysosomes; mitochondrial health; mitochondrial myopathies
  32. Mol Metab. 2021 Jun 08. pii: S2212-8778(21)00114-9. [Epub ahead of print] 101269
      OBJECTIVE: Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB).METHODS: Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies.
    RESULTS: Exogenously administered AcAc is reduced to D-βOHB, and increases hepatic NAD+/NADH ratio, reflecting hepatic BDH1 activity. Livers of hepatocyte-specific BDH1 deficient mice produced no D-βOHB, but due to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte specific BDH1 deficient mice showed diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal overall hepatic energy charge. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia.
    CONCLUSIONS: Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.
    Keywords:  Glucose Metabolism; Hepatic ketogenesis; Liver oxidative metabolism; Mitochondrial redox homeostasis; metabolic Flux; metabolomics
  33. Nat Commun. 2021 06 09. 12(1): 3486
      The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.
  34. Cancer Cell. 2021 May 28. pii: S1535-6108(21)00271-3. [Epub ahead of print]
      The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.
    Keywords:  CRISPR; EMT; S100; barcoding; epithelial-to-mesenchymal transition; evolving barcodes; ineage tracing; metastasis; phylogenetics; single cell
  35. Methods Mol Biol. 2021 ;2275 329-339
      Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain . Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria . In the assay, mitochondrial suspensions are spiked with CoQ10-[2H9] internal standard (IS), extracted with organic solvents and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).
    Keywords:  Coenzyme Q10; Isotope dilution; LC-MS/MS; Mitochondrial disease; Ubiquinone
  36. Autophagy. 2021 Jun 09. 1-3
      Spermidine is a natural polyamine, central to cellular homeostasis and growth, that promotes macroautophagy/autophagy. The polyamine pathway is highly conserved from bacteria to mammals and spermidine (prominently found in some kinds of aged cheese, wheat germs, nuts, soybeans, and fermented products thereof, among others) is an intrinsic part of the human diet. Apart from nutrition, spermidine is available to mammalian organisms from intracellular biosynthesis and microbial production in the gut. Importantly, externally supplied spermidine (via drinking water or food) prolongs lifespan, activates autophagy, improves mitochondrial function, and refills polyamine pools that decline during aging in various tissues of model organisms, including mice. In two adjacent studies, we explored how dietary spermidine supplementation enhances eEF5/EIF5A hypusination, cerebral mitochondrial function and cognition in aging Drosophila melanogaster and mice.
    Keywords:  Autophagy; Drosophila; Pink1; hypusination; learning; memory; mitophagy; polyamines; spermidine
  37. DNA Repair (Amst). 2021 May 11. pii: S1568-7864(21)00090-2. [Epub ahead of print]105 103134
      Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives.
    Keywords:  DNA damage; Formaldehyde; Mitochondrial DNA; One carbon metabolism
  38. Stem Cell Res Ther. 2021 Jun 05. 12(1): 320
      Immunotherapy has ushered in an exciting new era for cancer treatment. The recent discovery and success of immune checkpoint blockade and chimeric antigen receptor (CAR) T cell adoptive cell transfer has raised interest in using other immune cells, including Natural Killer (NK) cells, which might overcome some limitations with CAR T cell therapy. In this review article, we discuss the evidence that cellular metabolism is crucial for NK cell effector function. Additionally, potential strategies to optimise the metabolism of therapeutic NK cells for improved function within the metabolically adverse tumour microenvironment will be explored.
    Keywords:  Cancer immunotherapy; Metabolic signalling; Metabolism; Mitochondria; Natural Killer cells; Nutrients; PPAR; SREBP; Tumour microenvironment; cMyc; mTORC1
  39. Methods Mol Biol. 2021 ;2275 393-402
      Kidneys are highly aerobic organs and their function is tightly coupled to mitochondrial energy production. Renal tubular cells, particularly the proximal tubule (PT), require an abundance of mitochondria to provide sufficient energy for regulating fluid and electrolyte balance. Meanwhile, mitochondrial defects are implicated in a range of different kidney diseases. Multiphoton microscopy (MP) is a powerful tool that allows detailed study of mitochondrial morphology, dynamics, and function in kidney tissue. Here, we describe how MP can be used to image mitochondria in kidney tubular cells, either ex vivo in tissue slices or in vivo in living rodents, using both endogenous and exogenous fluorescent molecules. Moreover, changes in mitochondrial signals can be followed in real time in response to different insults or stimuli, in parallel with other important readouts of kidney tubular function, such as solute uptake and trafficking.
    Keywords:  Fluorescence imaging; Kidney; Mitochondria; Multiphoton microscopy
  40. Front Oncol. 2021 ;11 682911
      Cholesterol is a ubiquitous sterol with many biological functions, which are crucial for proper cellular signaling and physiology. Indeed, cholesterol is essential in maintaining membrane physical properties, while its metabolism is involved in bile acid production and steroid hormone biosynthesis. Additionally, isoprenoids metabolites of the mevalonate pathway support protein-prenylation and dolichol, ubiquinone and the heme a biosynthesis. Cancer cells rely on cholesterol to satisfy their increased nutrient demands and to support their uncontrolled growth, thus promoting tumor development and progression. Indeed, transformed cells reprogram cholesterol metabolism either by increasing its uptake and de novo biosynthesis, or deregulating the efflux. Alternatively, tumor can efficiently accumulate cholesterol into lipid droplets and deeply modify the activity of key cholesterol homeostasis regulators. In light of these considerations, altered pathways of cholesterol metabolism might represent intriguing pharmacological targets for the development of exploitable strategies in the context of cancer therapy. Thus, this work aims to discuss the emerging evidence of in vitro and in vivo studies, as well as clinical trials, on the role of cholesterol pathways in the treatment of cancer, starting from already available cholesterol-lowering drugs (statins or fibrates), and moving towards novel potential pharmacological inhibitors or selective target modulators.
    Keywords:  cancer; cancer therapy; cholesterol; metabolic reprogramming; metabolic targeting agents; pharmacological modulation; pharmacological targeting
  41. Front Mol Biosci. 2021 ;8 686412
      Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite with wide-ranging and significant roles in the cell. Defects in NAD+ metabolism have been associated with many human disorders; it is therefore an emerging therapeutic target. Moreover, NAD+ metabolism is perturbed during colonization by a variety of pathogens, either due to the molecular mechanisms employed by these infectious agents or by the host immune response they trigger. Three main biosynthetic pathways, including the de novo and salvage pathways, contribute to the production of NAD+ with a high degree of conservation from bacteria to humans. De novo biosynthesis, which begins with l-tryptophan in eukaryotes, is also known as the kynurenine pathway. Intermediates of this pathway have various beneficial and deleterious effects on cellular health in different contexts. For example, dysregulation of this pathway is linked to neurotoxicity and oxidative stress. Activation of the de novo pathway is also implicated in various infections and inflammatory signaling. Given the dynamic flexibility and multiple roles of NAD+ intermediates, it is important to understand the interconnections and cross-regulations of NAD+ precursors and associated signaling pathways to understand how cells regulate NAD+ homeostasis in response to various growth conditions. Although regulation of NAD+ homeostasis remains incompletely understood, studies in the genetically tractable budding yeast Saccharomyces cerevisiae may help provide some molecular basis for how NAD+ homeostasis factors contribute to the maintenance and regulation of cellular function and how they are regulated by various nutritional and stress signals. Here we present a brief overview of recent insights and discoveries made with respect to the relationship between NAD+ metabolism and selected human disorders and infections, with a particular focus on the de novo pathway. We also discuss how studies in budding yeast may help elucidate the regulation of NAD+ homeostasis.
    Keywords:  budding yeast; de novo NAD+ synthesis; immune response; infection; kynurenine metabolites; quinolinic acid; siutuin
  42. Nat Commun. 2021 06 08. 12(1): 3464
      Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.
  43. Front Cell Dev Biol. 2021 ;9 658861
      Colorectal cancer is the leading cause of death from cancer globally. The current treatment protocol still heavily relies on early detection and surgery. The molecular mechanisms underlying development of colorectal cancer are clinically important and determine the prognosis and treatment response. The arginine metabolism pathway is hyperactive in colorectal cancer and several molecules involved in the pathway are potential targets for chemoprevention and targeted colorectal cancer therapy. Endothelial nitric oxide synthase (eNOS), argininosuccinate synthetase and ornithine decarboxylase (ODC) are the main enzymes for arginine metabolism. Limiting arginine-rich meat consumption and inhibiting ODC activity largely reduces polyamine synthesis and the incidence of colorectal cancer. Arginine transporter CAT-1 and Human member 14 of the solute carrier family 6 (SLC6A14) are overexpressed in colorectal cancer cells and contributes to intracellular arginine levels. Human member 9 of the solute carrier family 38 (SLC38A9) serves as a component of the lysosomal arginine-sensing machinery. Pharmaceutical inhibition of single enzyme or arginine transporter is hard to meet requirement of restoring of abnormal arginine metabolic network. Apart from application in early screening for colorectal cancer, microRNA-based therapeutic strategy that simultaneously manipulating multiple targets involved in arginine metabolism brings promising future in the treatment of colorectal cancer.
    Keywords:  arginine metabolism; colorectal cancer; signal pathway; stem cells; transporters protein
  44. J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00525-1. [Epub ahead of print] 100736
      Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain (ETC) at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3-4 min at 37°C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool, blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and therefore, cellular energy metabolism, particularly during cell proliferation.
    Keywords:  Hydrogen sulfide; aerobic glycolysis; electron transport chain; sulfide quinone oxidoreductase