bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒11‒20
33 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Cancer Res Commun. 2022 Oct 10. 2(10): 1144-1161
      Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.Significance: ClpP agonists disrupt mitochondrial homeostasis by activating mitochondrial matrix protease ClpP. We report that ClpP agonists inhibit cell growth and cancer stem cell functions in breast cancer models by modulating multiple metabolic pathways essential to cancer stem cell function.
  2. Cancer Sci. 2022 Nov 16.
      Haplogroups and single nucleotide polymorphisms (SNP) of mitochondrial DNA (mtDNA) were associated with prognosis of many types of cancer patients. However, whether mtDNA haplogroups contribute to clinical outcomes of colorectal cancer (CRC) in Chinese population remains to be determined. In this study, mtDNA of tissue samples from 445 CRC patients from Northwestern China was sequenced to evaluate the association between haplogroup and prognosis. The mtDNA sequencing data of 1015 CRC patients from Southern China were collected for validation. We found patients with mtDNA haplogroup M7 had a significantly higher death risk when compared to patients with other haplogroups in both Northwestern (HR = 3.093, 95%CI = 1.768-5.411, P < 0.001) and Southern (HR = 1.607, 95%CI = 1.050-2.459, P = 0.029) China. Then, a haplogroup M7-based mtSNP classifier was selected by using LASSO Cox regression analysis. A nomogram comprising mtSNP classifier and clinicopathological variables was developed to predict prognosis of CRC patients (AUC 0.735, 95%CI = 0.679-0.791). Furthermore, patients with high- and low-risk scores calculated by the haplogroup M7-based mtSNP classifier exhibited significantly different overall survival (OS) and recurrence-free survival (RFS) (all P < 0.001). Finally, RNA-seq and immunohistochemical analyses indicated the poor prognosis of patients with haplogroup M7 may be related to mitochondrial dysfunction and immune abnormalities in CRC tissues. In conclusion, the haplogroup M7 and haplogroup M7-based mtSNP classifier seems to be a practical and reliable prognostic predictor for CRC patients, which provides a potential tool of clinical decision making for patients with haplogroup M7 in Chinese population.
    Keywords:  colorectal cancer; haplogroup; mitochondrial DNA; prognosis; single nucleotide polymorphisms
  3. Biochim Biophys Acta Bioenerg. 2022 Nov 14. pii: S0005-2728(22)00406-6. [Epub ahead of print] 148936
      Oxidative phosphorylation is a common process to most organisms in which the main function is to generate an electrochemical gradient across the inner mitochondrial membrane (IMM) and to make energy available to the cell. However, plants, many fungi and some animals maintain non-energy conserving oxidases which serve as a bypass to coupled respiration. Namely, the alternative NADH:ubiquinone oxidoreductase NDI1, present in the complex I (CI)-lacking Saccharomyces cerevisiae, and the alternative oxidase, ubiquinol:oxygen oxidoreductase AOX, present in many organisms across different kingdoms. In the last few years, these alternative oxidases have been used to dissect previously indivisible processes in bioenergetics and have helped to discover, understand, and corroborate important processes in mitochondria. Here, we review how the use of alternative oxidases have contributed to the knowledge in CI stability, bioenergetics, redox biology, and the implications of their use in current and future research.
    Keywords:  AOX; Alternative oxidase; CoQ pool; Oxphos; ROS
  4. Mol Cell Biol. 2022 Nov 14. e0014322
      Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.
    Keywords:  FAM210B; erythroid differentiation; mitochondria; mitochondrial ATP synthase
  5. Sci Immunol. 2022 Nov 25. 7(77): eabm8182
      T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.
  6. Cell Oncol (Dordr). 2022 Nov 19.
      BACKGROUND: Although isocitrate dehydrogenase 2 (IDH2) mutations have been the hotspots in recent anticancer studies, the impact of wild-type IDH2 on cancer cell growth and metabolic alterations is still elusive.METHODS: IDH2 expression in CRC tissues was evaluated by immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Cell functional assays included CCK8 and colony formation for cell proliferation in vitro and ectopic xenograft as in vivo experimental model for tumor progression. A targeted metabolomic procedure was performed by liquid chromatography/tandem mass spectrometry to profile the metabolites from glycolysis and tricarboxylic acid (TCA) cycle. Mitochondrial function was assessed by measuring cellular oxygen consumption (OCR) and mitochondrial membrane potential (ΔΨ). Confocal microscope analysis and Western blotting were applied to detect the expression of GLUT1 and NF-κB signaling. O-GlcNAcylation and the interaction of IDH2 with OGT were confirmed by co-immunoprecipitation, followed by Western blotting analysis.
    RESULTS: IDH2 protein was highly expressed in CRC tissues, and correlated with poor survival of CRC patients. Wild-type IDH2 promoted CRC cell growth in vitro and tumor progression in xenograft mice. Overexpression of wild-type IDH2 significantly increased glycolysis and TCA cycle metabolites, the ratios of NADH/NAD+ and ATP/ADP, OCR and mitochondrial membrane potential (ΔΨ) in CRC cells. Furthermore, α-KG activated NF-κB signaling to promote glucose uptake by upregulating GLUT1. Interesting, O-GlcNAcylation enhanced the protein half-time of IDH2 by inhibiting ubiquitin-mediated proteasome degradation. The O-GlcNAc transferase (OGT)-IDH2 axis promoted CRC progression.
    CONCLUSION: Wild-type IDH2 reprogrammed glucose metabolism and bioenergetic production via the NF-κB signaling pathway to promote CRC development and progression. O-GlcNAcylation of IDH2 elevated the stability of IDH2 protein. And the axis of OGT-IDH2 played an essential promotive role in tumor progression, suggesting a novel potential therapeutic strategy in CRC treatment.
    Keywords:  Glucose uptake; Isocitrate dehydrogenase 2; Mitochondrial bioenergetics; NF-κB signaling; O-GlcNAc transferase; O-GlcNAcylation; Redox status
  7. BMB Rep. 2022 Nov 16. pii: 5731. [Epub ahead of print]
      Epithelial-to-mesenchymal transition (EMT)-subtype gastric cancers have the worst prognosis due to their higher recurrence rate, higher probability of developing metastases and higher chemo-resistance compared to those of other molecular subtypes. Pharmacologically actionable somatic mutations are rarely found in EMT-subtype gastric cancers, limiting the utility of targeted therapies. Here, we conducted a high-throughput chemical screen using 37 gastric cancer cell lines and 48,467 synthetic small-molecule compounds. We identified YK-135, a small-molecule compound that showed higher cytotoxicity toward EMT-subtype gastric cancer cell lines than toward non-EMT-subtype gastric cancer cell lines. YK-135 exerts its cytotoxic effects by inhibiting mitochondrial complex I activity and inducing AMP-activated protein kinase (AMPK)-mediated apoptosis. We found that the lower glycolytic capacity of the EMT-subtype gastric cancer cells confers synthetic lethality to the inhibition of mitochondrial complex I, possibly by failing to maintain energy homeostasis. Other well-known mitochondrial complex I inhibitors (e.g., rotenone and phenformin) mimic the efficacy of YK-135, supporting our results. These findings highlight mitochondrial complex I inhibitors as promising therapeutic agents for EMT-subtype gastric cancers and YK-135 as a novel chemical scaffold for further drug development.
  8. Adv Sci (Weinh). 2022 Nov 16. e2202642
      Lacking a clear understanding of the molecular mechanism determining cancer cell sensitivity to oxidative phosphorylation (OXPHOS) inhibition limits the development of OXPHOS-targeting cancer treatment. Here, cancer cell lines sensitive or resistant to OXPHOS inhibition are identified by screening. OXPHOS inhibition-sensitive cancer cells possess increased OXPHOS activity and silenced nicotinamide N-methyltransferase (NNMT) expression. NNMT expression negatively correlates with OXPHOS inhibition sensitivity and functionally downregulates the intracellular levels of S-adenosyl methionine (SAM). Expression of DNA methyltransferase 1 (DNMT1), a SAM consumer, positively correlates with OXPHOS inhibition sensitivity. NNMT overexpression and DNMT1 inhibition render OXPHOS inhibition-sensitive cancer cells resistant. Importantly, treatments of OXPHOS inhibitors (Gboxin and Berberine) hamper the growth of mouse tumor xenografts by OXPHOS inhibition sensitive but not resistant cancer cells. What's more, the retrospective study of 62 tumor samples from a clinical trial demonstrates that administration of Berberine reduces the tumor recurrence rate of NNMTlow /DNMT1high but not NNMThigh /DNMT1low colorectal adenomas (CRAs). These results thus reveal a critical role of the NNMT-DNMT1 axis in determining cancer cell reliance on mitochondrial OXPHOS and suggest that NNMT and DNMT1 are faithful biomarkers for OXPHOS-targeting cancer therapies.
    Keywords:  DNA methylation; N-methyltransferase (NNMT); S-adenosyl methionine (SAM); biomarker; cancer; oxidative phosphorylation (OXPHOS)
  9. Life Sci Alliance. 2023 Jan;pii: e202201510. [Epub ahead of print]6(1):
      Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.
  10. Front Physiol. 2022 ;13 1004099
      Mitochondria contain their own DNA, mitochondrial DNA, which encodes thirteen proteins. However, mitochondria require thousands of proteins encoded in the nucleus to carry out their many functions. Identifying the definitive mitochondrial proteome has been challenging as methods isolating mitochondrial proteins differ and different tissues and organisms may have specialized proteomes. Mitochondrial diseases arising from single gene mutations in nucleus encoded genes could affect the mitochondrial proteome, but deciphering which effects are due to loss of specific pathways or to accumulated general mitochondrial damage is difficult. To identify specific versus general effects, we have taken advantage of mutations in three Drosophila genes, clueless, Sod2, and Pink1, which are required for mitochondrial function through different pathways. We measured changes in each mutant's mitochondrial proteome using quantitative tandem mass tag mass spectrometry. Our analysis identified protein classes that are unique to each mutant and those shared between them, suggesting that some changes in the mitochondrial proteome are due to general mitochondrial damage whereas others are gene specific. For example, clueless mutants had the greatest number of less and more abundant mitochondrial proteins whereas loss of all three genes increased stress and metabolism proteins. This study is the first to directly compare in vivo steady state levels of mitochondrial proteins by examining loss of three pathways critical for mitochondrial function. These data could be useful to understand disease etiology, and how mutations in genes critical for mitochondrial function cause specific mitochondrial proteomic changes as opposed to changes due to generalized mitochondrial damage.
    Keywords:  Clueless; PINK1; SOD2; drosophila; mitochondria; mitochondrial proteome; respiratory chain complexes
  11. Nat Commun. 2022 Nov 17. 13(1): 7047
      Chemotherapy can eradicate a majority of cancer cells. However, a small population of tumor cells often survives drug treatments through genetic and/or non-genetic mechanisms, leading to tumor recurrence. Here we report a reversible chemoresistance phenotype regulated by the mTOR pathway. Through a genome-wide CRISPR knockout library screen in pancreatic cancer cells treated with chemotherapeutic agents, we have identified the mTOR pathway as a prominent determinant of chemosensitivity. Pharmacological suppression of mTOR activity in cancer cells from diverse tissue origins leads to the persistence of a reversibly resistant population, which is otherwise eliminated by chemotherapeutic agents. Conversely, activation of the mTOR pathway increases chemosensitivity in vitro and in vivo and predicts better survival among various human cancers. Persister cells display a senescence phenotype. Inhibition of mTOR does not induce cellular senescence per se, but rather promotes the survival of senescent cells through regulation of autophagy and G2/M cell cycle arrest, as revealed by a small-molecule chemical library screen. Thus, mTOR plays a causal yet paradoxical role in regulating chemotherapeutic response; inhibition of the mTOR pathway, while suppressing tumor expansion, facilitates the development of a reversible drug-tolerant senescence state.
  12. Front Oncol. 2022 ;12 958155
      Human TRIAP1 (TP53-regulated inhibitor of apoptosis 1; also known as p53CSV for p53-inducible cell survival factor) is the homolog of yeast Mdm35, a well-known chaperone that interacts with the Ups/PRELI family proteins and participates in the intramitochondrial transfer of lipids for the synthesis of cardiolipin (CL) and phosphatidylethanolamine. Although recent reports indicate that TRIAP1 is a prosurvival factor abnormally overexpressed in various types of cancer, knowledge about its molecular and metabolic function in human cells is still elusive. It is therefore critical to understand the metabolic and proliferative advantages that TRIAP1 expression provides to cancer cells. Here, in a colorectal cancer cell model, we report that the expression of TRIAP1 supports cancer cell proliferation and tumorigenesis. Depletion of TRIAP1 perturbed the mitochondrial ultrastructure, without a major impact on CL levels and mitochondrial activity. TRIAP1 depletion caused extramitochondrial perturbations resulting in changes in the endoplasmic reticulum-dependent lipid homeostasis and induction of a p53-mediated stress response. Furthermore, we observed that TRIAP1 depletion conferred a robust p53-mediated resistance to the metabolic stress caused by glutamine deprivation. These findings highlight the importance of TRIAP1 in tumorigenesis and indicate that the loss of TRIAP1 has extramitochondrial consequences that could impact on the metabolic plasticity of cancer cells and their response to conditions of nutrient deprivation.
    Keywords:  TRIAP1/Mdm35; coiled-coil-helix-coiled-coil-helix domain (CHCHD)-containing proteins; colon cancer; glutamine starvation; lipid homeostasis; mitochondria; p53-mediated stress response
  13. Cell Death Differ. 2022 Nov 14.
      How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.
  14. Proc Natl Acad Sci U S A. 2022 Nov 22. 119(47): e2210730119
      Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.
    Keywords:  ATAD3A; Drp1; mitochondrial fission; mtDNA nucleoid; respiratory complex
  15. Cancer Res. 2022 Nov 15. 82(22): 4124-4125
      The role of exercise in cancer progression is an emerging field of research, with intriguing evidence for physical activity playing an inhibitory role in cancer onset. In their recent publication, Sheinboim and colleagues demonstrate the impact of physical exercise on melanoma primary tumor growth and metastasis. They establish that physical exercise decreases metastatic spread, using both human epidemiologic data and in vivo models of melanoma metastasis. Systemic metabolic reprogramming of organs, induced by exercise, leads to a decrease in melanoma growth and progression as healthy organs are able to outcompete melanoma cells for nutrients. Exercise led to systemic metabolic changes in carbohydrate metabolism, glycolysis, and oxidative phosphorylation as well as mitochondrial biogenesis. Interestingly, the "metabolic shield" created by exercise could be reversed using the mTOR inhibitor rapamycin. This study highlights the importance of metabolic plasticity in metastasis and uncovers a direct link between systemic metabolic reprogramming and mTOR signaling. Overall, the study by Sheinboim and colleagues provides a more detailed understanding of the metastatic requirements in the context of energy and nutrient availability and the impact of exercise on cancer progression, highlighting novel opportunities for therapeutic intervention. See related article by Sheinboim et al., p. 4164.
  16. Cell Mol Biol Lett. 2022 Nov 18. 27(1): 100
      BACKGROUND: Metformin is an inhibitor of oxidative phosphorylation that displays an array of anticancer activities. The interference of metformin with the activity of multi-drug resistance systems in cancer cells has been reported. However, the consequences of the acquired chemoresistance for the adaptative responses of cancer cells to metformin-induced stress and for their phenotypic evolution remain unaddressed.METHODS: Using a range of phenotypic and metabolic assays, we assessed the sensitivity of human prostate cancer PC-3 and DU145 cells, and their drug-resistant lineages (PC-3_DCX20 and DU145_DCX20), to combined docetaxel/metformin stress. Their adaptation responses have been assessed, in particular the shifts in their metabolic profile and invasiveness.
    RESULTS: Metformin increased the sensitivity of PC-3 wild-type (WT) cells to docetaxel, as illustrated by the attenuation of their motility, proliferation, and viability after the combined drug application. These effects correlated with the accumulation of energy carriers (NAD(P)H and ATP) and with the inactivation of ABC drug transporters in docetaxel/metformin-treated PC-3 WT cells. Both PC-3 WT and PC-3_DCX20 reacted to metformin with the Warburg effect; however, PC-3_DCX20 cells were considerably less susceptible to the cytostatic/misbalancing effects of metformin. Concomitantly, an epithelial-mesenchymal transition and Cx43 upregulation was seen in these cells, but not in other more docetaxel/metformin-sensitive DU145_DCX20 populations. Stronger cytostatic effects of the combined fenofibrate/docetaxel treatment confirmed that the fine-tuning of the balance between energy supply and expenditure determines cellular welfare under metabolic stress.
    CONCLUSIONS: Collectively, our data identify the mechanisms that underlie the limited potential of metformin for the chemotherapy of drug-resistant tumors. Metformin can enhance the sensitivity of cancer cells to chemotherapy by inducing their metabolic decoupling/imbalance. However, the acquired chemoresistance of cancer cells impairs this effect, facilitates cellular adaptation to metabolic stress, and prompts the invasive front formation.
    Keywords:  Drug resistance; Metabolic adaptation; Metabolic stress; Metformin; Prostate cancer
  17. Cell Biosci. 2022 Nov 12. 12(1): 183
      BACKGROUND: Exploiting cancer metabolism during nutrient availability holds immense potential for the clinical and therapeutic benefits of hepatocellular carcinoma (HCC) patients. Dietary methionine is a metabolic dependence of cancer development, but how the signal transduction integrates methionine status to achieve the physiological demand of cancer cells remains unknown.METHODS: Low or high levels of dietary methionine was fed to mouse models with patient-derived xenograft or diethyl-nitrosamine induced liver cancer. RNA sequence and metabolomics were performed to reveal the profound effect of methionine restriction on gene expression and metabolite changes. Immunostaining, sphere formation assays, in vivo tumourigenicity, migration and self-renewal ability were conducted to demonstrate the efficacy of methionine restriction and sorafenib.
    RESULTS: We discovered that mTORC1-c-Myc-SIRT4 axis was abnormally regulated in a methionine-dependent manner and affected the HCC progression. c-Myc rewires methionine metabolism through TRIM32 mediated degradation of SIRT4, which regulates MAT2A activity by ADP-ribosylation on amino acid residue glutamic acid 111. MAT2A is a key enzyme to generate S-adenosylmethionine (SAM). Loss of SIRT4 activates MAT2A, thereby increasing SAM level and dynamically regulating gene expression, which triggers the high proliferation rate of tumour cells. SIRT4 exerts its tumour suppressive function with targeted therapy (sorafenib) by affecting methionine, redox and nucleotide metabolism.
    CONCLUSIONS: These findings establish a novel characterization of the signaling transduction and the metabolic consequences of dietary methionine restriction in malignant liver tissue of mice. mTORC1, c-Myc, SIRT4 and ADP ribosylation site of MAT2A are promising clinical and therapeutic targets for the HCC treatment.
    Keywords:  ADP ribosylation; Cancer; MAT2A; Methionine metabolism; SIRT4; TRIM32; c-Myc; mTOR
  18. Nat Metab. 2022 Nov 14.
      The tumour microenvironment possesses mechanisms that suppress anti-tumour immunity. Itaconate is a metabolite produced from the Krebs cycle intermediate cis-aconitate by the activity of immune-responsive gene 1 (IRG1). While it is known to be immune modulatory, the role of itaconate in anti-tumour immunity is unclear. Here, we demonstrate that myeloid-derived suppressor cells (MDSCs) secrete itaconate that can be taken up by CD8+ T cells and suppress their proliferation, cytokine production and cytolytic activity. Metabolite profiling, stable-isotope tracing and metabolite supplementation studies indicated that itaconate suppressed the biosynthesis of aspartate and serine/glycine in CD8+ T cells to attenuate their proliferation and function. Host deletion of Irg1 in female mice bearing allografted tumours resulted in decreased tumour growth, inhibited the immune-suppressive activities of MDSCs, promoted anti-tumour immunity of CD8+ T cells and enhanced the anti-tumour activity of anti-PD-1 antibody treatment. Furthermore, we found a significant negative correlation between IRG1 expression and response to PD-1 immune checkpoint blockade in patients with melanoma. Our findings not only reveal a previously unknown role of itaconate as an immune checkpoint metabolite secreted from MDSCs to suppress CD8+ T cells, but also establish IRG1 as a myeloid-selective target in immunometabolism whose inhibition promotes anti-tumour immunity and enhances the efficacy of immune checkpoint protein blockade.
  19. iScience. 2022 Nov 18. 25(11): 105447
      An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.
    Keywords:  Cell biology; Functional aspects of cell biology; Molecular biology
  20. iScience. 2022 Nov 18. 25(11): 105458
      mTORC1 is aberrantly activated in cancer and in the genetic tumor syndrome tuberous sclerosis complex (TSC), which is caused by loss-of-function mutations in the TSC complex, a negative regulator of mTORC1. Clinically approved mTORC1 inhibitors, such as rapamycin, elicit a cytostatic effect that fails to eliminate tumors and is rapidly reversible. We sought to determine the effects of mTORC1 on the core regulators of intrinsic apoptosis. In TSC2-deficient cells and tumors, we find that mTORC1 inhibitors shift cellular dependence from MCL-1 to BCL-2 and BCL-XL for survival, thereby altering susceptibility to BH3 mimetics that target specific pro-survival BCL-2 proteins. The BCL-2/BCL-XL inhibitor ABT-263 synergizes with rapamycin to induce apoptosis in TSC-deficient cells and in a mouse tumor model of TSC, resulting in a more complete and durable response. These data expose a therapeutic vulnerability in regulation of the apoptotic machinery downstream of mTORC1 that promotes a cytotoxic response to rapamycin.
    Keywords:  Biological sciences; Cancer; Cell biology; Molecular biology
  21. Cell Signal. 2022 Nov 12. pii: S0898-6568(22)00286-8. [Epub ahead of print] 110524
      Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.
    Keywords:  EF-Tu(mt) (TUFM); Fyn and c-Src kinases; Mitochondrial protein synthesis; SFKs; Translation; Tyr phosphorylation
  22. Leukemia. 2022 Nov 18.
      Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.
  23. Sci Adv. 2022 Nov 16. 8(46): eabq5234
      A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.
  24. Sci Rep. 2022 Nov 18. 12(1): 19841
      The phenomenon of intercellular mitochondrial transfer has attracted great attention in various fields of research, including stem cell biology. Elucidating the mechanism of mitochondrial transfer from healthy stem cells to cells with mitochondrial dysfunction may lead to the development of novel stem cell therapies to treat mitochondrial diseases, among other advances. To visually evaluate and analyze the mitochondrial transfer process, dual fluorescent labeling systems are often used to distinguish the mitochondria of donor and recipient cells. Although enhanced green fluorescent protein (EGFP) has been well-characterized for labeling mitochondria, other colors of fluorescent protein have been less extensively evaluated in the context of mitochondrial transfer. Here, we generated different lentiviral vectors with mitochondria-targeted red fluorescent proteins (RFPs), including DsRed, mCherry (both from Discosoma sp.) Kusabira orange (mKOκ, from Verrillofungia concinna), and TurboRFP (from Entacmaea quadricolor). Among these proteins, mitochondria-targeted DsRed and its variant mCherry often generated bright aggregates in the lysosome while other proteins did not. We further validated that TurboRFP-labeled mitochondria were successfully transferred from amniotic epithelial cells, one of the candidates for donor stem cells, to mitochondria-damaged recipient cells without losing the membrane potential. Our study provides new insight into the genetic labeling of mitochondria with red fluorescent proteins, which may be utilized to analyze the mechanism of intercellular mitochondrial transfer.
  25. Biochim Biophys Acta Bioenerg. 2022 Nov 12. pii: S0005-2728(22)00404-2. [Epub ahead of print] 148934
      The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420-450 nm), alpha (560-630 nm) and red (630-700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or of apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.
    Keywords:  Cytochrome c oxidase; Membrane potential; Mitochondria; Respiration; Spectroscopy; electron transfer
  26. J Biol Chem. 2022 Nov 12. pii: S0021-9258(22)01147-4. [Epub ahead of print] 102704
      The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.
    Keywords:  MAP1LC3B/LC3B; PHB2; Parkin; mitophagy; ubiquitination
  27. Biochem Biophys Res Commun. 2022 Nov 11. pii: S0006-291X(22)01562-5. [Epub ahead of print]637 144-152
      Cancer cells exhibit increased glutamine consumption compared to normal cells, supporting cell survival and proliferation. Glutamine is converted to α-ketoglutarate (αKG), which then enters the tricarboxylic acid cycle to generate ATP. Recently, therapeutic modulation of glutamine metabolism has become an attractive metabolic anti-cancer strategy. However, how synergistic combination therapy is required to overcome glutamine metabolism drug resistance remains elusive. To address this issue, we first investigated the role of αKG in regulating gene expression in several cancer cell lines. Using RNA-seq analysis and histone modification screening, we demonstrated that αKG reduced the expression of the immediate early gene (IEG) in cancer cells in an H3K27 acetylation-dependent manner. Conversely, glutaminase (GLS) inhibitors induce IEG expression in cancer cells. Furthermore, we showed that siRNA knockdown of orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) induces IEG expression. Notably, the NR4A1 agonist cytosporone B sensitizes GLS inhibitor resistance to cancer cell death. Together, these findings indicate that therapeutic targeting of IEG dysregulation by αKG can be a potentially effective anti-cancer therapeutic strategy for glutamine metabolism inhibitors.
    Keywords:  Cancer cells; GLS inhibitor; IEG; NR4A; αKG
  28. Mol Cell. 2022 Nov 17. pii: S1097-2765(22)01055-3. [Epub ahead of print]82(22): 4246-4261.e11
      Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.
    Keywords:  E2A; HCC; TEAD2; acetyl-CoA metabolism; dedifferentiation; hepatocellular carcinoma; protein acetylation; transcriptional reprogramming
  29. Nature. 2022 Nov 16.
      Solid cancers exhibit a dynamic balance between cell death and proliferation ensuring continuous tumour maintenance and growth1,2. Increasing evidence links enhanced cancer cell apoptosis to paracrine activation of cells in the tumour microenvironment initiating tissue repair programs that support tumour growth3,4, yet the direct effects of dying cancer cells on neighbouring tumour epithelia and how this paracrine effect potentially contributes to therapy resistance are unclear. Here we demonstrate that chemotherapy-induced tumour cell death in patient-derived colorectal tumour organoids causes ATP release triggering P2X4 (also known as P2RX4) to mediate an mTOR-dependent pro-survival program in neighbouring cancer cells, which renders surviving tumour epithelia sensitive to mTOR inhibition. The induced mTOR addiction in persisting epithelial cells is due to elevated production of reactive oxygen species and subsequent increased DNA damage in response to the death of neighbouring cells. Accordingly, inhibition of the P2X4 receptor or direct mTOR blockade prevents induction of S6 phosphorylation and synergizes with chemotherapy to cause massive cell death induced by reactive oxygen species and marked tumour regression that is not seen when individually applied. Conversely, scavenging of reactive oxygen species prevents cancer cells from becoming reliant on mTOR activation. Collectively, our findings show that dying cancer cells establish a new dependency on anti-apoptotic programs in their surviving neighbours, thereby creating an opportunity for combination therapy in P2X4-expressing epithelial tumours.
  30. Sci Adv. 2022 Nov 18. 8(46): eadd3855
      Respiratory complex I is a ~1-MDa proton pump in mitochondria. Its structure has been revealed in great detail, but the structural basis of its assembly, in humans involving at least 15 assembly factors, is essentially unknown. We determined cryo-electron microscopy structures of assembly intermediates associated with assembly factor NDUFAF1 in a yeast model system. Subunits ND2 and NDUFC2 together with assembly factors NDUFAF1 and CIA84 form the nucleation point of the NDUFAF1-dependent assembly pathway. Unexpectedly, the cardiolipin remodeling enzyme tafazzin is an integral component of this core complex. In a later intermediate, all 12 subunits of the proximal proton pump module have assembled. NDUFAF1 locks the central ND3 subunit in an assembly-competent conformation, and major rearrangements of central subunits are required for complex I maturation.
  31. Mater Today Bio. 2022 Dec 15. 17 100475
      Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human-induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under conditions of fluidic flow with the same precision and relevance as current standard static cell cultures.
    Keywords:  Extracellular flux; Human stem cells; Metabolism; Organ-on-Chip; Oxygen; Sensors; pH
  32. Nat Commun. 2022 Nov 17. 13(1): 7031
      An enhanced NADH/NAD+ ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD+ ratio and meantime reduce the NADPH/NADP+ ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD+ ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD+ and NADPH/NADP+.