bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒03‒13
39 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Mol Cancer Ther. 2022 Feb 28. pii: molcanther.0925.2021. [Epub ahead of print]
      Cancer cells undergo significant "metabolic remodeling" to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer (CRC) cells, ATP is produced by mitochondrial oxidative phosphorylation (OXPHOS) and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in CRC cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit CRC cell growth in vitro and in vivo. This study suggests that an electron transport chain (ETC) inhibitor (i.e. DBI-1) and a glucose transport inhibitor, (i.e. BAY-876) are potentially effective combination for CRC treatment.
  2. RNA. 2022 Mar 07. pii: rna.079097.122. [Epub ahead of print]
      Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.
    Keywords:  FACS; FUNCAT; HPG; Mitochondria; Translation
  3. Front Cell Dev Biol. 2022 ;10 781558
      Mitochondria are biosynthetic, bioenergetic, and signaling organelles with a critical role in cellular physiology. Dysfunctional mitochondria are associated with aging and underlie the cause of a wide range of diseases, from neurodegeneration to cancer. Through signaling, mitochondria regulate diverse biological outcomes. The maintenance of the mitochondrial membrane potential, for instance, is essential for proliferation, the release of mitochondrial reactive oxygen species, and oxygen sensing. The loss of mitochondrial membrane potential triggers pathways to clear damaged mitochondria and often results in cell death. In this study, we conducted a genome-wide positive selection CRISPR screen using a combination of mitochondrial inhibitors to uncover genes involved in sustaining a mitochondrial membrane potential, and therefore avoid cell death when the electron transport chain is impaired. Our screen identified genes involved in mitochondrial protein translation and ATP synthesis as essential for the induction of cell death when cells lose their mitochondrial membrane potential. This report intends to provide potential targets for the treatment of diseases associated with mitochondrial dysfunction.
    Keywords:  ATP synthase; CRISPR screen; cell death; mitochondria; mitochondrial membrane potential; mitochondrial protein translation
  4. Nature. 2022 Mar 09.
      The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.
  5. Cells. 2022 Mar 02. pii: 862. [Epub ahead of print]11(5):
      Aging is the greatest challenge to humankind worldwide. Aging is associated with a progressive loss of physiological integrity due to a decline in cellular metabolism and functions. Such metabolic changes lead to age-related diseases, thereby compromising human health for the remaining life. Thus, there is an urgent need to identify geroprotectors that regulate metabolic functions to target the aging biological processes. Nutrients are the major regulator of metabolic activities to coordinate cell growth and development. Iron is an important nutrient involved in several biological functions, including metabolism. In this study using yeast as an aging model organism, we show that iron supplementation delays aging and increases the cellular lifespan. To determine how iron supplementation increases lifespan, we performed a gene expression analysis of mitochondria, the main cellular hub of iron utilization. Quantitative analysis of gene expression data reveals that iron supplementation upregulates the expression of the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) genes. Furthermore, in agreement with the expression profiles of mitochondrial genes, ATP level is elevated by iron supplementation, which is required for increasing the cellular lifespan. To confirm, we tested the role of iron supplementation in the AMPK knockout mutant. AMPK is a highly conserved controller of mitochondrial metabolism and energy homeostasis. Remarkably, iron supplementation rescued the short lifespan of the AMPK knockout mutant and confirmed its anti-aging role through the enhancement of mitochondrial functions. Thus, our results suggest a potential therapeutic use of iron supplementation to delay aging and prolong healthspan.
    Keywords:  AMPK; Saccharomyces cerevisiae; cellular lifespan extension; chronological aging; iron; mitochondria
  6. Cell Rep. 2022 Mar 08. pii: S2211-1247(22)00226-1. [Epub ahead of print]38(10): 110493
      Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.
    Keywords:  CRISPR-Cas9 screen; acidosis; oxidative phosphorylation; tumor acidity
  7. J Biol Chem. 2022 Mar 02. pii: S0021-9258(22)00231-9. [Epub ahead of print] 101791
      Many proteins are modified by post-translational methylation, introduced by a number of methyltransferases (MTases). Protein methylation plays important roles in modulating protein function, and thus in optimizing and regulating cellular and physiological processes. Research has mainly focused on nuclear and cytosolic protein methylation, but it has been known for many years that also mitochondrial proteins are methylated. During the last decade, significant progress has been made on identifying the MTases responsible for mitochondrial protein methylation and addressing its functional significance. In particular, several novel human MTases have been uncovered that methylate lysine, arginine, histidine, and glutamine residues in various mitochondrial substrates. Several of these substrates are key components of the bioenergetics machinery, e.g. respiratory Complex I, citrate synthase and the ATP synthase. In the present review we report the status of the field of mitochondrial protein methylation, with a particular emphasis on recently discovered human MTases. We also discuss evolutionary aspects and functional significance of mitochondrial protein methylation, and present an outlook for this emergent research field.
    Keywords:  ATP synthase; bioenergetics; electron transport chain; methyltransferase; mitochondria; oxidative phosphorylation; protein methylation
  8. Arch Biochem Biophys. 2022 Mar 08. pii: S0003-9861(22)00057-1. [Epub ahead of print] 109172
      Mitochondria change their morphology and inner membrane structure depending on their activity. Since mitochondrial activity also depends on their structure, it is important to elucidate the interrelationship between the activity and structure of mitochondria. However, the mechanism by which mitochondrial activity affects the structure of cristae, the folded structure of the inner membrane, is not well understood. In this study, the effect of the mitochondrial activity on the cristae structure was investigated by examining the structural rigidity of cristae. Taking advantage of the fact that unfolding of cristae induces mitochondrial swelling, we investigated the relationship between mitochondrial activity and the susceptibility to swelling. The swelling of individual isolated mitochondria exposed to a hypotonic solution was observed with an optical microscope. The presence of respiratory substrates (malate and glutamate) increased the percentage of mitochondria that underwent swelling, and the further addition of rotenone or KCN (inhibitors of proton pumps) reversed the increase. In the absence of respiratory substrates, acidification of the buffer surrounding the mitochondria also increased the percentage of swollen mitochondria. These observations suggest that acidification of the outer surface of inner membranes, especially intracristal space, by proton translocation from the matrix to the intracristal space, decreases the structural rigidity of the cristae. This interpretation was verified by the observation that ADP or CCCP, which induces proton re-entry to the matrix, suppressed the mitochondrial swelling in the presence of respiratory substrates. The addition of CCCP to the cells induced a morphological change in mitochondria from an initial elongated structure to a largely curved structure at pH 7.4, but there were no morphological changes when the pH of the cytosol dropped to 6.2. These results suggest that a low pH in the intracristal space may be helpful in maintaining the elongated structure of mitochondria. The present study shows that proton pumping by the electron transfer chain is the mechanism underlying mitochondrial morphology and the flexibility of cristae structure.
    Keywords:  Inner mitochondrial membrane; Intracristal space; Proton pumps; mitochondria; mitochondrial cristae; mitochondrial swelling
  9. Front Aging. 2022 ;pii: 805126. [Epub ahead of print]2
      Mitochondria are the main source of energy used to maintain cellular homeostasis. This aspect of mitochondrial biology underlies their putative role in age-associated tissue dysfunction. Proper functioning of the electron transport chain (ETC), which is partially encoded by the extra-nuclear mitochondrial genome (mtDNA), is key to maintaining this energy production. The acquisition of de novo somatic mutations that interrupt the function of the ETC have long been associated with aging and common diseases of the elderly. Yet, despite over 30 years of study, the exact role(s) mtDNA mutations play in driving aging and its associated pathologies remains under considerable debate. Furthermore, even fundamental aspects of age-related mtDNA mutagenesis, such as when mutations arise during aging, where and how often they occur across tissues, and the specific mechanisms that give rise to them, remain poorly understood. In this review, we address the current understanding of the somatic mtDNA mutations, with an emphasis of when, where, and how these mutations arise during aging. Additionally, we highlight current limitations in our knowledge and critically evaluate the controversies stemming from these limitations. Lastly, we highlight new and emerging technologies that offer potential ways forward in increasing our understanding of somatic mtDNA mutagenesis in the aging process.
    Keywords:  aging; mitochondria; mtDNA; mutagenesis; sequencing; somatic mutations
  10. Anal Biochem. 2022 Mar 05. pii: S0003-2697(22)00102-6. [Epub ahead of print] 114646
      Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD+ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis. Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 105 complex I molecules/cell. Our method can be used for accurate quantification of complex I content.
    Keywords:  Astrocytes; Flavin mononucleotide; Fluorescence; Mitochondrial complex I; Respiratory chain
  11. Int J Mol Sci. 2022 Mar 07. pii: 2888. [Epub ahead of print]23(5):
      Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.
    Keywords:  cancer cell lines; cellular proliferation; mitochondrial electron transfer chain; mitochondrial unfolded protein response; molecular hydrogen
  12. Autophagy. 2022 Mar 08. 1-18
      Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.
    Keywords:  ATG4B; biosynthesis; cell death; electron transport chain; endothelial cells; mitochondria; oxidative phosphorylation; oxidative stress; reactive oxygen species
  13. Cancers (Basel). 2022 Feb 22. pii: 1108. [Epub ahead of print]14(5):
      The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6), in the metabolic adaptation of basal-like (AnxA6-low) versus mesenchymal-like (AnxA6-high), as well as in lapatinib-resistant TNBC cells. Using model basal-like and mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. The downregulation of AnxA6 in TNBC cells generally attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity resulting in a quiescent metabolic phenotype. We also show that AnxA6 depletion in mesenchymal-like TNBC cells was associated with a rapid uptake and mitochondrial fatty acid oxidation and diminished lipid droplet accumulation and altered the lipogenic metabolic phenotype of these cells to a lypolytic metabolic phenotype. The overexpression or chronic lapatinib-induced upregulation of AnxA6 in AnxA6-low TNBC cells reversed the quiescent/lypolytic phenotype to a more lipogenic/glycolytic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies distinct metabolic adaptations of basal-like and mesenchymal-like TNBC subsets in response to cellular stress and/or therapeutic intervention and suggest AnxA6 as a biomarker for metabolic subtyping of TNBC subsets.
    Keywords:  Annexin A6; breast cancer; lipid metabolism; metabolic reprogramming; tyrosine kinase inhibitors
  14. Redox Biol. 2022 Feb 24. pii: S2213-2317(22)00040-4. [Epub ahead of print]51 102268
      mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing. In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia. These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.
    Keywords:  G6PD; ROS; T-ALL; mTOR
  15. Cancers (Basel). 2022 Feb 27. pii: 1230. [Epub ahead of print]14(5):
      Cancer is one of the leading causes of mortality worldwide. Beyond standard therapeutic options, whose effectiveness is often reduced by drug resistance, repurposing of the antidiabetic drug metformin appears promising. Heme metabolism plays a pivotal role in the control of metabolic adaptations that sustain cancer cell proliferation. Recently, we demonstrated the existence of a functional axis between the heme synthetic enzyme ALAS1 and the heme exporter FLVCR1a exploited by cancer cells to down-modulate oxidative metabolism. In colorectal cancer cell lines, the inhibition of heme synthesis-export system was associated with reduced proliferation and survival. Here, we aim to assess whether the inhibition of the heme synthesis-export system affects the sensitivity of colorectal cancer cells to metformin. Our data demonstrate that the inhibition of this system, either by blocking heme efflux with a FLVCR1a specific shRNA or by inhibiting heme synthesis with 5-aminolevulinic acid, improves metformin anti-proliferative effect on colorectal cancer cell lines. In addition, we demonstrated that the same effect can be obtained in other kinds of cancer cell lines. Our study provides an in vitro proof of concept of the possibility to target heme metabolism in association with metformin to counteract cancer cell growth.
    Keywords:  ALAS1; FLVCR1; FLVCR1a; cancer; heme; metabolism; metformin; mitochondria; therapy
  16. J Biol Chem. 2022 Mar 04. pii: S0021-9258(22)00215-0. [Epub ahead of print] 101775
      It's widely accepted that increasing mitochondrial respiration plays a pivotal role during osteoclastogenesis. Mitochondrial pyruvate carrier (MPC) is the key transporter that links glycolysis to mitochondrial respiration but little is known about its role during osteoclastogenesis. Our goal was to determine the effects of its blockade on osteoclastogenesis and bone resorption in vivo and in vitro. To address this issue, we performed gene knockdown or pharmacologically inhibited MPC in primary bone marrow-derived monocytes (BMMs) or in an ovariectomized mouse model. We also studied the metabolic changes in RANKL-induced differentiating BMMs with MPC blockade and performed rescue experiments. We found that MPC blockade resulted in decreased osteoclastogenesis both in vivo and in vitro and inhibiting MPC significantly alleviated ovariectomy-induced trabecular bone loss. Further investigations showed that MPC blockade significantly reversed the metabolic profile related to RANK activation, including decreased intermediates involved in citric acid cycle and glutamine metabolism. Moreover, metabolic flux analysis verified that MPC blockade decreased pyruvate flux into TCA cycle with no significant effect on glycolysis. Besides, MPC blockade resulted in suppressed mitochondrial biogenesis in addition to oxidative phosphorylation. Rescue experiments revealed that inhibiting pyruvate dehydrogenase kinase (PDK) via sodium dichloroacetate (DCA), but not targeting glutamine metabolism, could reverse the effects of MPC blockade on osteoclastogenesis. These implied that the effects of MPC blockade were mediated by reduced pyruvate fuel into citric acid cycle in multiple aspects. Taken together, our data demonstrated the inhibitory effects of MPC blockade on osteoclastogenesis, which was mediated by decreased mitochondrial energy production.
    Keywords:  energy production; mitochondrial biogenesis; mitochondrial pyruvate carrier; osteoclast; osteoporosis
  17. Nutrients. 2022 Jan 27. pii: 559. [Epub ahead of print]14(3):
      The fatty acid elongase elongation of very long-chain fatty acids protein 2 (ELOVL2) controls the elongation of polyunsaturated fatty acids (PUFA) producing precursors for omega-3, docosahexaenoic acid (DHA), and omega-6, docosapentaenoic acid (DPAn-6) in vivo. Expectedly, Elovl2-ablation drastically reduced the DHA and DPAn-6 in liver mitochondrial membranes. Unexpectedly, however, total PUFAs levels decreased further than could be explained by Elovl2 ablation. The lipid peroxidation process was not involved in PUFAs reduction since malondialdehyde-lysine (MDAL) and other oxidative stress biomarkers were not enhanced. The content of mitochondrial respiratory chain proteins remained unchanged. Still, membrane remodeling was associated with the high voltage-dependent anion channel (VDAC) and adenine nucleotide translocase 2 (ANT2), a possible reflection of the increased demand on phospholipid transport to the mitochondria. Mitochondrial function was impaired despite preserved content of the respiratory chain proteins and the absence of oxidative damage. Oligomycin-insensitive oxygen consumption increased, and coefficients of respiratory control were reduced by 50%. The mitochondria became very sensitive to fatty acid-induced uncoupling and permeabilization, where ANT2 is involved. Mitochondrial volume and number of peroxisomes increased as revealed by transmission electron microscopy. In conclusion, the results imply that endogenous DHA production is vital for the normal function of mouse liver mitochondria and could be relevant not only for mice but also for human metabolism.
    Keywords:  adenine nucleotide translocase; docosahexaenoic acid (DHA) deficiency; membrane permeabilization; mitochondrial function; oxidative damage markers; polyunsaturated fatty acids
  18. Cells. 2022 Feb 23. pii: 771. [Epub ahead of print]11(5):
      Mitochondria are subcellular organelles that are a hub for key biological processes, such as bioenergetic, biosynthetic, and signaling functions. Mitochondria are implicated in all oncogenic processes, from malignant transformation to metastasis and resistance to chemotherapeutics. The harsh tumor environment constantly exposes cancer cells to cytotoxic stressors, such as nutrient starvation, low oxygen, and oxidative stress. Excessive or prolonged exposure to these stressors can cause irreversible mitochondrial damage, leading to cell death. To survive hostile microenvironments that perturb mitochondrial function, cancer cells activate a stress response to maintain mitochondrial protein and genome integrity. This adaptive mechanism, which is closely linked to mitochondrial function, enables rapid adjustment and survival in harsh environmental conditions encountered during tumor dissemination, thereby promoting cancer progression. In this review, we describe how the mitochondria stress response contributes to the acquisition of typical malignant traits and highlight the potential of targeting the mitochondrial stress response as an anti-cancer therapeutic strategy.
    Keywords:  mitochondrial dynamics; mitochondrial protein quality control; mitochondrial stress response; mitophagy; mtDNA
  19. Cancers (Basel). 2022 Mar 05. pii: 1343. [Epub ahead of print]14(5):
      Metformin and 2-deoxy-D-glucose (2DG) exhibit multiple metabolic and immunomodulatory anti-cancer effects, such as suppressed proliferation or PD-L1 expression. Their combination or 2DG alone induce triple-negative breast cancer (TNBC) cell detachment, but their effects on mitochondria, crucial for anchorage-independent growth and metastasis formation, have not yet been evaluated. In the present study, we explored the effects of metformin, 2DG and their combination (metformin + 2DG) on TNBC cell mitochondria in vitro. Metformin + 2DG increased mitochondrial mass in TNBC cells. This was associated with an increased size but not number of morphologically normal mitochondria and driven by the induction of mitochondrial biogenesis rather than suppressed mitophagy. 2DG and metformin + 2DG strongly induced the unfolded protein response by inhibiting protein N-glycosylation. Together with adequate energy stress, this was one of the possible triggers of mitochondrial enlargement. Suppressed N-glycosylation by 2DG or metformin + 2DG also caused PD-L1 deglycosylation and reduced surface expression in MDA-MB-231 cells. PD-L1 was increased in low glucose and normalized by both drugs. 2DG and metformin + 2DG reduced PD-1 expression in Jurkat cells beyond the effects on activation, while cytokine secretion was mostly preserved. Despite increasing mitochondrial mass in TNBC cells, metformin and 2DG could therefore potentially be used as an adjunct therapy to improve anti-tumor immunity in TNBC.
    Keywords:  2-deoxy-D-glucose; AMPK; ER stress; PD-1/PD-L1 axis; T cells; anchorage-independence; metformin; mitochondrial biogenesis; protein N-glycosylation; triple-negative breast cancer
  20. Cell Rep. 2022 Mar 08. pii: S2211-1247(22)00208-X. [Epub ahead of print]38(10): 110475
      Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated. Here, in CHCHD10 knockin mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses, leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.
    Keywords:  1C metabolism; CHCHD10; coiled-coil-helix-coiled-coil-helix domain containing 10; heart, cardiomyopathy; heme; integrated stress response; metabolic rewiring; mitochondria
  21. Curr Protoc. 2022 Mar;2(3): e390
      The mitochondrion is a key intracellular organelle regulating metabolic processes, oxidative stress, energy production, calcium homeostasis, and cell survival. Protein phosphorylation plays an important role in regulating mitochondrial functions and cellular signaling pathways. Dysregulation of protein phosphorylation status can cause protein malfunction and abnormal signal transduction, leading to organ dysfunction and disease. Investigating the mitochondrial phosphoproteins is therefore crucial to better understand the molecular and pathogenic mechanisms of many metabolic disorders. Conventional analyses of phosphoproteins, for instance, via western blotting, can be done only for proteins for which specific antibodies to their phosphorylated forms are available. Moreover, such an approach is not suitable for large-scale study of phosphoproteins. Currently, proteomics represents an important tool for large-scale analysis of proteins and their post-translational modifications, including phosphorylation. Here, we provide step-by-step protocols for the proteomics analysis of mitochondrial phosphoproteins (the phosphoproteome), using renal tubular cells as an example. These protocols include methods to effectively isolate mitochondria and to validate the efficacy of mitochondrial enrichment as well as its purity. We also provide detailed protocols for performing both gel-based and gel-free phosphoproteome analyses. The gel-based analysis involves two-dimensional gel electrophoresis and phosphoprotein-specific staining, followed by protein identification via mass spectrometry, whereas the gel-free approach is based on in-solution mass spectrometric identification of specific phosphorylation sites and residues. In all, these approaches allow large-scale analyses of mitochondrial phosphoproteins that can be applied to other cells and tissues of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Mitochondrial isolation/purification from renal tubular cells Support Protocol: Validation of enrichment efficacy and purity of mitochondrial isolation Basic Protocol 2: Gel-based phosphoproteome analysis Basic Protocol 3: Gel-free phosphoproteome analysis.
    Keywords:  PTMs ; mass spectrometry; mitochondria; phosphoproteome; phosphorylation; proteome; proteomics
  22. Physiol Rep. 2022 Mar;10(5): e15151
      The global prevalence of type 2 diabetes (T2D) is expected to exceed 642 million people by 2040. Metformin is a widely used biguanide T2D therapy, associated with rare but serious events of lactic acidosis, in particular with predisposing conditions (e.g., renal failure or major surgery). Imeglimin, a recently approved drug, is the first in a new class (novel mode of action) of T2D medicines. Although not a biguanide, Imeglimin shares a chemical moiety with Metformin and also modulates mitochondrial complex I activity, a potential mechanism for Metformin-mediated lactate accumulation. We interrogated the potential for Imeglimin to induce lacticacidosis in relevant animal models and further assessed differences in key mechanisms known for Metformin's effects. In a dog model of major surgery, Metformin or Imeglimin (30-1000 mg/kg) was acutely administered, only Metformin-induced lactate accumulation and pH decrease leading to lactic acidosis with fatality at the highest dose. Rats with gentamycin-induced renal insufficiency received Metformin or Imeglimin (50-100 mg/kg/h), only Metformin increased lactatemia and H+ concentrations with mortality at higher doses. Plasma levels of Metformin and Imeglimin were similar in both models. Mice were chronically treated with Metformin or Imeglimin 200 mg/kg bid. Only Metformin produced hyperlactatemia after acute intraperitoneal glucose loading. Ex vivo measurements revealed higher mitochondrial complex I inhibition with Metformin versus slight effects with Imeglimin. Another mechanism implicated in Metformin's effects on lactate production was assessed: in isolated rat, liver mitochondria exposed to Imeglimin or Metformin, only Metformin (50-250 µM) inhibited the mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH). In liver samples from chronically treated mice, measured mGPDH activity was lower with Metformin versus Imeglimin. These data indicate that the risk of lactic acidosis with Imeglimin treatment may be lower than with Metformin and confirm that the underlying mechanisms of action are distinct, supporting its potential utility for patients with predisposing conditions.
    Keywords:  Imeglimin; Metformin; complex I; lactic acidosis; mGPDH
  23. Mol Oncol. 2022 Mar 12.
      Metabolic dysregulation is an important hallmark of cancer. Nicotinamide (NAM), a water-soluble amide form of niacin (vitamin B3), is currently available as a supplement for maintaining general physiologic functions. NAM is a crucial regulator of mitochondrial metabolism and redox reactions. In this study, we aimed to identify the mechanistic link between NAM-induced metabolic regulation and the therapeutic efficacy of NAM in triple-negative breast cancer (TNBC). The combined analysis using multi-omics systems biology showed that NAM decreased mitochondrial membrane potential and ATP production but increased the activities of reverse electron transport (RET), fatty acid β-oxidation, and glycerophospholipid/sphingolipid metabolic pathways in TNBC, collectively leading to an increase in the levels of reactive oxygen species (ROS). The increased ROS levels triggered apoptosis and suppressed tumor growth and metastasis of TNBC in both human organoids and xenograft mouse models. Our results showed that NAM treatment leads to cancer cell death in TNBC via mitochondrial dysfunction and activation of ROS by bifurcating metabolic pathways (RET and lipid metabolism); this provides insights into the repositioning of NAM supplement as a next-generation anti-metabolic agent for TNBC treatment.
    Keywords:  Metabolism; Nicotinamide; Organoids; Proteogenomics; Reactive Oxygen Species; Triple Negative Breast Neoplasms
  24. Front Oncol. 2022 ;12 783908
      The grade of malignancy differs among cancer cell types, yet it remains the burden of genetic studies to understand the reasons behind this observation. Metabolic studies of cancer, based on the Warburg effect or aerobic glycolysis, have also not provided any clarity. Instead, the significance of oxidative phosphorylation (OXPHOS) has been found to play critical roles in aggressive cancer cells. In this perspective, metabolic symbiosis is addressed as one of the ultimate causes of the grade of cancer malignancy. Metabolic symbiosis gives rise to metabolic heterogeneities which enable cancer cells to acquire greater opportunities for proliferation and metastasis in tumor microenvironments. This study introduces a real-time new imaging technique to visualize metabolic symbiosis between cancer-associated fibroblasts (CAFs) and cancer cells based on the metabolic oscillations in these cells. The causality of cellular oscillations in cancer cells and CAFs, connected through lactate transport, is a key point for the development of this novel technique.
    Keywords:  cancer; heterogeneity; malignancy; metabolic oscillations; symbiosis
  25. Curr Opin Physiol. 2021 Dec;pii: 100487. [Epub ahead of print]24
      Mitochondria and lipid droplets in the insulin resistant skeletal muscle of type 2 diabetic individuals have both been heavily investigated independently and are characterized by more fragmented, dysfunctional mitochondrial networks and larger lipid droplets compared to skeletal muscle of healthy individuals. Specialized contacts between mitochondrial and lipid droplet membranes are known to decrease in diabetic muscle, though it remains unclear how energy transfer at the remaining mitochondria-lipid droplet contact sites may be altered by type 2 diabetes. The purpose of this review is to highlight recent data on mitochondrial structure and function and lipid droplet dynamics in type 2 diabetic skeletal muscle and to underscore the need for more detailed investigations into the functional nature of mitochondria-lipid droplet interactions in type 2 diabetes.
    Keywords:  Mitochondria; diabetes; insulin resistance; lipid droplets; mitochondrial network
  26. Nat Commun. 2022 Mar 07. 13(1): 1199
      Deregulation of the BCL-2 family interaction network ensures cancer resistance to apoptosis and is a major challenge to current treatments. Cancer cells commonly evade apoptosis through upregulation of the BCL-2 anti-apoptotic proteins; however, more resistant cancers also downregulate or inactivate pro-apoptotic proteins to suppress apoptosis. Here, we find that apoptosis resistance in a diverse panel of solid and hematological malignancies is mediated by both overexpression of BCL-XL and an unprimed apoptotic state, limiting direct and indirect activation mechanisms of pro-apoptotic BAX. Both survival mechanisms can be overcome by the combination of an orally bioavailable BAX activator, BTSA1.2 with Navitoclax. The combination demonstrates synergistic efficacy in apoptosis-resistant cancer cells, xenografts, and patient-derived tumors while sparing healthy tissues. Additionally, functional assays and genomic markers are identified to predict sensitive tumors to the combination treatment. These findings advance the understanding of apoptosis resistance mechanisms and demonstrate a novel therapeutic strategy for cancer treatment.
  27. J Lipid Res. 2022 Mar 02. pii: S0022-2275(22)00021-9. [Epub ahead of print] 100188
      Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.
    Keywords:  CPT; click; fatty acid metabolism; lipid oxidation; lipid tracer; lipidomics; mid-chain fatty acid; mitochondria; peroxisomes; β-oxidation
  28. J Biol Chem. 2022 Mar 02. pii: S0021-9258(22)00228-9. [Epub ahead of print] 101788
      A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m3C) modifications. In mammals, the number of tRNAs containing m3C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m3C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m3C. We demonstrate that human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells could be rescued by re-expression of wildtype METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential function for m3C modification in mitochondrial tRNA structure.
    Keywords:  3-methylcytosine; METTL8; m(3)C; mitochondria; tRNA; tRNA modification
  29. Int J Mol Sci. 2022 Feb 27. pii: 2627. [Epub ahead of print]23(5):
      In the past decade, anti-tumour immune responses have been successfully exploited to improve the outcome of patients with different cancers. Significant progress has been made in taking advantage of different types of T cell functions for therapeutic purposes. Despite these achievements, only a subset of patients respond favorably to immunotherapy. Therefore, there is a need of novel approaches to improve the effector functions of immune cells and to recognize the major targets of anti-tumour immunity. A major hallmark of cancer is metabolic rewiring associated with switch of mitochondrial functions. These changes are a consequence of high energy demand and increased macromolecular synthesis in cancer cells. Such adaptations in tumour cells might generate novel targets of tumour therapy, including the generation of neoantigens. Here, we review the most recent advances in research on the immune response to mitochondrial proteins in different cellular conditions.
    Keywords:  T cell response; cancer neoantigens; mitochondria; mtDNA mutations; post translational modifications
  30. Bioorg Chem. 2022 Feb 28. pii: S0045-2068(22)00116-X. [Epub ahead of print]122 105711
      Overexpression of thioredoxin reductase (TrxR) has been linked to tumorigenesis and phenotypic maintenance of malignant tumors. Thus, targeting TrxR with natural molecules is a promising strategy for developing anticancer drugs. Sinomenine is a naturally occurring alkaloid isolated from Sinomenium acutum. The drug, Zhengqing Fengtongning made from sinomenine, has been universally applied in rheumatoid arthritis treatment in China as well as other Asian countries for decades. Recently, increasing evidence indicates that sinomenine appears to be a promising therapeutic agent against various cancer cells. However, the exact mechanism underlying the anticancer activity of sinomenine remains unclear. In this study, we identified sinomenine as a kind of new inhibitor for TrxR. Pharmacological inhibition of TrxR by sinomenine results in the decrease of thiols content, increases the levels of reactive oxygen species, and finally facilitates oxidative stress-mediated cancer cell apoptosis. It is vital that knockdown in TrxR1 by shRNA can increase cell sensitivity to sinomenine. Treatment with sinomenine in vivo leads to a decrease in TrxR activity and tumor growth, and an increase in apoptosis. Our findings provide a novel action mechanism related to sinomenine and presents an insight on how to develop sinomenine as a chemotherapeutic agent for cancer therapy.
    Keywords:  Apoptosis; Chemotherapeutic agents; Oxidative stress; Reactive oxygen species; Sinomenine; Thioredoxin reductase
  31. Cancer Res. 2022 Mar 03. pii: canres.2778.2021. [Epub ahead of print]
      The dynamic composition of the tumor microenvironment (TME) can markedly alter the response to targeted therapies for colorectal cancer (CRC). Cancer associated fibroblasts (CAF) are major components of TMEs that can direct and induce infiltration of immunosuppressive cells through secreted cytokines such as CXCL12. Ketogenic diets (KD) can inhibit tumor growth and enhance the anticancer effects of immune checkpoint blockade. However, the role of ketogenesis on the immunosuppressive TME is not known. Here, we show that decreased ketogenesis is a signature of CRC and that an increase in ketogenesis using a KD decreases CXCL12 production in tumors, serum, liver, and lungs. Moreover, increasing ketogenesis by overexpression of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) or treatment with the ketone body β-hydroxybutyrate markedly decreased expression of KLF5, which binds the CXCL12 promoter and induces CXCL12 expression in CAFs. KD decreased intratumoral accumulation of immunosuppressive cells, increased infiltration of NK and cytotoxic T cells, and enhanced the anticancer effects of PD-1 blockade in murine-derived CRC. Furthermore, increasing ketogenesis inhibited CRC migration, invasion, and metastasis in vitro and in vivo. Overall, ketogenesis is downregulated in the CRC TME, and increased ketogenesis represses KLF5-dependent CXCL12 expression to improve the immunosuppressive TME, which leads to the enhanced efficacy of immunotherapy and reduced metastasis. Importantly, this work demonstrates that downregulation of de novo ketogenesis in the TME is a critical step in CRC progression.
  32. J Transl Med. 2022 Mar 10. 20(1): 118
      BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways.METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself.
    RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT.
    CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.
    Keywords:  BRAF oncogenic signaling; MAPK; NAMPT; NAMPT inhibitors; Oncogene; Transformation
  33. Nature. 2022 Mar 09.
      Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.
  34. Redox Biol. 2022 Feb 18. pii: S2213-2317(22)00044-1. [Epub ahead of print]51 102272
      Cutaneous melanoma is the deadliest type of skin cancer, although it accounts for a minority of all skin cancers. Oxidative stress is involved in all stages of melanomagenesis and cutaneous melanoma can sustain a much higher load of Reactive Oxygen Species (ROS) than normal tissues. Melanoma cells exploit specific antioxidant machinery to support redox homeostasis. The enzyme UBIA prenyltransferase domain-containing protein 1 (UBIAD1) is responsible for the biosynthesis of non-mitochondrial CoQ10 and plays an important role as antioxidant enzyme. Whether UBIAD1 is involved in melanoma progression has not been addressed, yet. Here, we provide evidence that UBIAD1 expression is associated with poor overall survival (OS) in human melanoma patients. Furthermore, UBIAD1 and CoQ10 levels are upregulated in melanoma cells with respect to melanocytes. We show that UBIAD1 and plasma membrane CoQ10 sustain melanoma cell survival and proliferation by preventing lipid peroxidation and cell death. Additionally, we show that the NAD(P)H Quinone Dehydrogenase 1 (NQO1), responsible for the 2-electron reduction of CoQ10 on plasma membranes, acts downstream of UBIAD1 to support melanoma survival. By showing that the CoQ10-producing enzyme UBIAD1 counteracts oxidative stress and lipid peroxidation events in cutaneous melanoma, this work may open to new therapeutic investigations based on UBIAD1/CoQ10 loss to cure melanoma.
    Keywords:  Antioxidant response; CoQ10; Lipid peroxidation; Melanoma; NQO1; UBIAD1
  35. Cancer Res. 2022 Feb 28. pii: canres.CAN-21-3983-E.2021. [Epub ahead of print]
      Metabolic reprogramming is a hallmark of malignant transformation, and loss of isozyme diversity (LID) contributes to this process. Isozymes are distinct proteins that catalyze the same enzymatic reaction but can have different kinetic characteristics, subcellular localization, and tissue specificity. Cancer-dominant isozymes that catalyze rate-limiting reactions in critical metabolic processes represent potential therapeutic targets. Here we examined the isozyme expression patterns of 1,319 enzymatic reactions in 14 cancer types and their matching normal tissues using TCGA mRNA expression data to identify isozymes that become cancer dominant. Of the reactions analyzed, 357 demonstrated LID in at least one cancer type. Assessment of the expression patterns in over 600 cell lines in the cancer cell line encyclopedia showed that these reactions reflect cellular changes instead of differences in tissue composition; 50% of the LID-affected isozymes showed cancer-dominant expression in the corresponding cell lines. The functional importance of the cancer-dominant isozymes was assessed in genome-wide CRISPR and RNAi loss-of-function screens: 17% were critical for cell proliferation, indicating their potential as therapeutic targets. Lists of prioritized novel metabolic targets were developed for 14 cancer types; the most broadly shared and functionally validated target was acetyl-CoA carboxylase-1 (ACC1). Small molecule inhibition of ACC reduced breast cancer viability in vitro and suppressed tumor growth in cell line- and patient-derived xenografts in vivo. Evaluation of the effects of drug treatment revealed significant metabolic and transcriptional perturbations. Overall, this systematic analysis of isozyme expression patterns elucidates an important aspect of cancer metabolic plasticity and reveals putative metabolic vulnerabilities.
  36. Sci Rep. 2022 Mar 09. 12(1): 3822
      Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase, which regulates multiple biological processes, including genome maintenance, aging, tumor suppression, and metabolism. While a number of substrates involved in these processes have been identified, the global landscape of the SIRT2 acetylome remains unclear. Using a label-free quantitative proteomic approach following enrichment for acetylated peptides from SIRT2-depleted and SIRT2-overexpressing HCT116 human colorectal cancer cells, we identified a total of 2,846 unique acetylation sites from 1414 proteins. 896 sites from 610 proteins showed a > 1.5-fold increase in acetylation with SIRT2 knockdown, and 509 sites from 361 proteins showed a > 1.5-fold decrease in acetylation with SIRT2 overexpression, with 184 proteins meeting both criteria. Sequence motif analyses identified several site-specific consensus sequence motifs preferentially recognized by SIRT2, most commonly KxxxxK(ac). Gene Ontology, KEGG, and MetaCore pathway analyses identified SIRT2 substrates involved in diverse pathways, including carbon metabolism, glycolysis, spliceosome, RNA transport, RNA binding, transcription, DNA damage response, the cell cycle, and colorectal cancer. Collectively, our findings expand on the number of known acetylation sites, substrates, and cellular pathways targeted by SIRT2, providing support for SIRT2 in regulating networks of proteins in diverse pathways and opening new avenues of investigation into SIRT2 function.
  37. STAR Protoc. 2022 Mar 18. 3(1): 101218
      Organoids can enable the study of solid tumors initiated from a single cancer stem cell (CSC) ex vivo. We describe a serial tumor organoid plating protocol using primary colorectal cancer (CRC) tissues as a rapid and cost-efficient approach to evaluate the impact of therapeutic interventions on CSC functions. We detail the isolation of primary colorectal CSCs, organoid embedding, serial passaging, and CSC-related analytical techniques. For complete details on the use and execution of this protocol, please refer to Masibag et al. (2021) and Bergin et al. (2021).
    Keywords:  Cancer; Cell isolation; Cell-based Assays; Health Sciences; Microscopy; Organoids; Single Cell; Stem Cells; Tissue Engineering
  38. J R Soc Interface. 2022 Mar;19(188): 20210883
      Many biological processes discriminate between correct and incorrect substrates through the kinetic proofreading mechanism that enables lower error at the cost of higher energy dissipation. Elucidating physico-chemical constraints for global minimization of dissipation and error is important for understanding enzyme evolution. Here, we identify theoretically a fundamental error-cost bound that tightly constrains the performance of proofreading networks under any parameter variations preserving the rate discrimination between substrates. The bound is kinetically controlled, i.e. completely determined by the difference between the transition state energies on the underlying free energy landscape. The importance of the bound is analysed for three biological processes. DNA replication by T7 DNA polymerase is shown to be nearly optimized, i.e. its kinetic parameters place it in the immediate proximity of the error-cost bound. The isoleucyl-tRNA synthetase (IleRS) of E. coli also operates close to the bound, but further optimization is prevented by the need for reaction speed. In contrast, E. coli ribosome operates in a high-dissipation regime, potentially in order to speed up protein production. Together, these findings establish a fundamental error-dissipation relation in biological proofreading networks and provide a theoretical framework for studying error-dissipation trade-off in other systems with biological discrimination.
    Keywords:  biochemical networks; cellular energetics; energy dissipation; kinetic proofreading; non-equilibrium thermodynamics; reaction kinetics and dynamics
  39. Cancer Lett. 2022 Mar 03. pii: S0304-3835(22)00091-X. [Epub ahead of print]534 215616
      Breast cancer is considered the most common malignancy, with the profound ability to perform a wide range of metabolic reprogramming. Within the breast cancer microenvironment, highly available cancer-associated adipocytes interact with cancer cells by releasing various adipocytokines and metabolites. Obesity is also an important factor in this manner, where the accumulation of adipose tissue next to tumor tissue is linked to the increased incidence, progression, and metastasis of breast cancer. The metabolic changes caused by the crosstalk between breast cancer cells and dysfunctional adipose tissue include glucose, lipid, and amino acid metabolism. Thus, preventing this interaction between breast cancer cells and dysfunctional adipose tissue might develop a promising therapeutic strategy against breast cancer. This review focused on the metabolic changes responsible for inducing the crosstalk between breast cancer cells and adipocytes. We also reviewed the recent updates in therapeutics designed to disrupt this interaction.
    Keywords:  Breast carcinoma; Cancer-associated adipocytes; Metabolic reprogramming; Obesity; Tumor microenvironment