bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒01‒10
fifty papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Nat Immunol. 2021 Jan 04.
      Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.
  2. Biochim Biophys Acta Bioenerg. 2021 Jan 04. pii: S0005-2728(20)30217-6. [Epub ahead of print] 148367
      The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.
    Keywords:  Exchange protein directly activated by cAMP (Epac); Glycolysis; NADH/NAD(+) redox state; Oxidative phosphorylation; Protein kinase a; Soluble adenylyl cyclase
  3. Front Oncol. 2020 ;10 604143
      The combined derangements in mitochondria network, function and dynamics can affect metabolism and ATP production, redox homeostasis and apoptosis triggering, contributing to cancer development in many different complex ways. In hematological malignancies, there is a strong relationship between cellular metabolism, mitochondrial bioenergetics, interconnections with supportive microenvironment and drug resistance. Lymphoma and chronic lymphocytic leukemia cells, e.g., adapt to intrinsic oxidative stress by increasing mitochondrial biogenesis. In other hematological disorders such as myeloma, on the contrary, bioenergetics changes, associated to increased mitochondrial fitness, derive from the adaptive response to drug-induced stress. In the bone marrow niche, a reverse Warburg effect has been recently described, consisting in metabolic changes occurring in stromal cells in the attempt to metabolically support adjacent cancer cells. Moreover, a physiological dynamic, based on mitochondria transfer, between tumor cells and their supporting stromal microenvironment has been described to sustain oxidative stress associated to proteostasis maintenance in multiple myeloma and leukemia. Increased mitochondrial biogenesis of tumor cells associated to acquisition of new mitochondria transferred by mesenchymal stromal cells results in augmented ATP production through increased oxidative phosphorylation (OX-PHOS), higher drug resistance, and resurgence after treatment. Accordingly, targeting mitochondrial biogenesis, electron transfer, mitochondrial DNA replication, or mitochondrial fatty acid transport increases therapy efficacy. In this review, we summarize selected examples of the mitochondrial derangements in hematological malignancies, which provide metabolic adaptation and apoptosis resistance, also supported by the crosstalk with tumor microenvironment. This field promises a rational design to improve target-therapy including the metabolic phenotype.
    Keywords:  OX-PHOS; acute myeloid leukemia; chronic lymphatic leukemia; lymphoma; mitochondria; multiple myeloma
  4. Cell Metab. 2021 Jan 05. pii: S1550-4131(20)30663-X. [Epub ahead of print]33(1): 128-144.e9
      The metabolic role of micropeptides generated from untranslated regions remains unclear. Here we describe MP31, a micropeptide encoded by the upstream open reading frame (uORF) of phosphatase and tensin homolog (PTEN) acting as a "circuit breaker" that limits lactate-pyruvate conversion in mitochondria by competing with mitochondrial lactate dehydrogenase (mLDH) for nicotinamide adenine dinucleotide (NAD+). Knocking out the MP31 homolog in mice enhanced global lactate metabolism, manifesting as accelerated oxidative phosphorylation (OXPHOS) and increased lactate consumption and production. Conditional knockout (cKO) of MP31 homolog in mouse astrocytes initiated gliomagenesis and shortened the overall survival of the animals, establishing a tumor-suppressing role for MP31. Recombinant MP31 administered intraperitoneally penetrated the blood-brain barrier and inhibited mice GBM xenografts without neurological toxicity, suggesting the clinical implication and application of this micropeptide. Our findings reveal a novel mode of MP31-orchestrated lactate metabolism reprogramming in glioblastoma.
    Keywords:  LDH; MP31; OXPHOS; PTEN; glioblastoma; lactate oxidation; tumorigenesis; uORF
  5. Int J Mol Sci. 2021 Jan 03. pii: E424. [Epub ahead of print]22(1):
      Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.
    Keywords:  HepG2 cells; NV118; cell-permeable succinate; mitochondria; platelets; statins
  6. J Ovarian Res. 2021 Jan 08. 14(1): 8
      Metabolic reprogramming refers to the transformation of the whole metabolic network including glycolysis and mitochondrial metabolism, mainly manifested in Warburg effect and mitochondrial metabolic reprogramming. The roles of miR-145 in glycolysis have been established in ovarian cancer cells. Howerer, its roles in mitochondrial metabolic reprogramming are still unclear. This study aims to identify whether miR-145 regulates mitochondrial metabolic reprogramming in ovarian cancer cells. First, functional experiment showed that overexpression of miR-145 inhibited mitochondrial function in ovarian cancer cells, evident by the decreased mtDNA copy numbers, ATP level, mitochondrial membrane potential, and the expression levels of mitochondrial markers. Mechanistically, miR-145 inhibited mitochondrial function by targeting ARL5B directly. Futhermore, miR-145 overexpression decreased ARL5B expression in ovarian cancer tissue subcutaneous tumors of nude mice. In conclusion, we have highlighted that miR-145 inhibits mitochondrial function and achieves this by targeting ARL5B directly for the first time. The results provides a more adequate theoretical basis for understanding the molecular pathology of ovarian cancer, and provides the necessary basic data for miR-145 as a potential diagnosis and treatment target for ovarian cancer.
    Keywords:  ARL5B; Mitochondrial function; Ovarian cancer; miR-145
  7. Front Cell Dev Biol. 2020 ;8 603688
      Arginyltransferase 1 (ATE1) is an evolutionary-conserved eukaryotic protein that localizes to the cytosol and nucleus. It is the only known enzyme in metazoans and fungi that catalyzes posttranslational arginylation. Lack of arginylation has been linked to an array of human disorders, including cancer, by altering the response to stress and the regulation of metabolism and apoptosis. Although mitochondria play relevant roles in these processes in health and disease, a causal relationship between ATE1 activity and mitochondrial biology has yet to be established. Here, we report a phylogenetic analysis that traces the roots of ATE1 to alpha-proteobacteria, the mitochondrion microbial ancestor. We then demonstrate that a small fraction of ATE1 localizes within mitochondria. Furthermore, the absence of ATE1 influences the levels, organization, and function of respiratory chain complexes in mouse cells. Specifically, ATE1-KO mouse embryonic fibroblasts have increased levels of respiratory supercomplexes I+III2+IVn. However, they have decreased mitochondrial respiration owing to severely lowered complex II levels, which leads to accumulation of succinate and downstream metabolic effects. Taken together, our findings establish a novel pathway for mitochondrial function regulation that might explain ATE1-dependent effects in various disease conditions, including cancer and aging, in which metabolic shifts are part of the pathogenic or deleterious underlying mechanism.
    Keywords:  arginylation; arginyltransferase; biogenesis; mitochondria; respiration; respiratory chain complexes
  8. J Clin Invest. 2021 Jan 04. pii: 133081. [Epub ahead of print]131(1):
      Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX+) mitochondrial respiration and low (OX-) mitochondrial respiration. The OX+ tumors poorly incorporated [18F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX+ tumor growth in vivo. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [18F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas.
    Keywords:  Bioenergetics; Metabolism; Oncology
  9. Redox Biol. 2020 Dec 24. pii: S2213-2317(20)31051-X. [Epub ahead of print] 101846
      Pharmacological targeting of mitochondrial ion channels is emerging as a promising approach to eliminate cancer cells; as most of these channels are differentially expressed and/or regulated in cancer cells in comparison to healthy ones, this strategy may selectively eliminate the former. Perturbation of ion fluxes across the outer and inner membranes is linked to alterations of redox state, membrane potential and bioenergetic efficiency. This leads to indirect modulation of oxidative phosphorylation, which is/may be fundamental for both cancer and cancer stem cell survival. Furthermore, given the crucial contribution of mitochondria to intrinsic apoptosis, modulation of their ion channels leading to cytochrome c release may be of great advantage in case of resistance to drugs triggering apoptotic events upstream of the mitochondrial phase. In the present review, we give an overview of the known mitochondrial ion channels and of their modulators capable of killing cancer cells. In addition, we discuss state-of-the-art strategies using mitochondriotropic drugs or peptide-based approaches allowing a more efficient and selective targeting of mitochondrial ion channel-linked events.
    Keywords:  Cancer; Channel interactions; Drug targeting; Ion channels; Mitochondria
  10. Int J Biol Sci. 2021 ;17(1): 285-297
      Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target.
    Keywords:  CD147; Melanoma; Mitochondria; aerobic oxidation; invasion
  11. Nat Commun. 2021 Jan 08. 12(1): 174
      The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.
  12. Cell Metab. 2020 Dec 24. pii: S1550-4131(20)30661-6. [Epub ahead of print]
      Platelets are known to enhance the wound-healing activity of mesenchymal stem cells (MSCs). However, the mechanism by which platelets improve the therapeutic potential of MSCs has not been elucidated. Here, we provide evidence that, upon their activation, platelets transfer respiratory-competent mitochondria to MSCs primarily via dynamin-dependent clathrin-mediated endocytosis. We found that this process enhances the therapeutic efficacy of MSCs following their engraftment in several mouse models of tissue injury, including full-thickness cutaneous wound and dystrophic skeletal muscle. By combining in vitro and in vivo experiments, we demonstrate that platelet-derived mitochondria promote the pro-angiogenic activity of MSCs via their metabolic remodeling. Notably, we show that activation of the de novo fatty acid synthesis pathway is required for increased secretion of pro-angiogenic factors by platelet-preconditioned MSCs. These results reveal a new mechanism by which platelets potentiate MSC properties and underline the importance of testing platelet mitochondria quality prior to their clinical use.
    Keywords:  angiogenesis; cell therapy; citrate; de novo; fatty acid synthesis; intercellular mitochondria transfer; mesenchymal stem cells; metabolism reprogramming; mitochondria; mitochondrial respiration; platelets
  13. Ann Surg Oncol. 2021 Jan 03.
      BACKGROUND: Anticancer drugs generate excessive reactive oxygen species (ROS), which can cause cell death. Cancer cells can resist this oxidative stress, but the mechanism of resistance and associations with chemoresistance are unclear. Here, we focused on Sirtuin 3 (SIRT3), a deacetylating mitochondrial enzyme, in oxidative stress resistance in colorectal cancer (CRC).METHODS: To evaluate SIRT3-related changes in mitochondrial function, ROS (mtROS) induction, and apoptosis, we used the human CRC cell lines HT29 and HCT116 transfected with short-hairpin RNA targeting SIRT3 and small interfering RNAs targeting superoxide dismutase 2 mitochondrial (SOD2) and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α). In 142 clinical specimens from patients with CRC, we also assessed the association of SIRT3 protein levels (high/low) and prognosis.
    RESULTS: SIRT3 expression correlated with mtROS generation and apoptosis induction in cells treated with anticancer agents. Suppressing SIRT3 increased mtROS levels and cell sensitivity to anticancer agents. SIRT3 knockdown decreased SOD2 expression and activity, and suppressing SOD2 also improved sensitivity to anticancer drugs. In addition, SIRT3 was recruited with PGC-1α under oxidative stress, and suppressing SIRT3 decreased PGC-1α expression and mitochondrial function. PGC-1α knockdown decreased mitochondrial activity and increased apoptosis in cells treated with anticancer drugs. In resected CRC specimens, high vs low SIRT3 protein levels were associated with significantly reduced cancer-specific survival.
    CONCLUSIONS: SIRT3 expression affected CRC cell chemoresistance through SOD2 and PGC-1α regulation and was an independent prognostic factor in CRC. SIRT3 may be a novel target for CRC therapies and a predictive marker of sensitivity to chemotherapy.
  14. Methods Mol Biol. 2021 ;2261 411-419
      In-depth analysis of the mitochondrial proteome can be greatly improved by analyzing isolated mitochondria instead of whole cells. However, isolation of sufficient amounts of mitochondria from cell culture has proven to be notoriously difficult due to small sample size. Thus, we have developed a reproducible, controllable, and highly customizable method to isolate high microgram to low milligram amounts of intact mitochondria from cell culture samples along with an optional density gradient purification. This chapter provides a methodological update of our approach and underlines the excellent quality and coverage of the mitochondrial proteome of crude and purified mitochondria from cultured liver cancer cell lines.
    Keywords:  Balch homogenizer; Cell culture; Mitochondria; Proteomics
  15. In Vivo. 2021 Jan-Feb;35(1):35(1): 341-348
      BACKGROUND/AIM: We investigated the hypothesis that dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and metformin (MET), an antidiabetic agent and complex I inhibitor, have synergistic cytotoxic effects in glioblastoma cells in vitro and in vivo.MATERIALS AND METHODS: We performed dose response experiments and combination index calculation. Apoptotic and necrotic cells were estimated by flow cytometry. Cell metabolism was evaluated by Seahorse analysis and lactate export. Overall survival and tumor volume growth experiments were performed in C57BL/6 mice GL-261 allograft model.
    RESULTS: DCA and MET showed dose-dependent cytotoxicity and synergistic effects. DCA alleviated the increase in lactate production induced by MET. Seahorse analysis showed that DCA treatment results in increased oxygen consumption rate, which is decreased by MET. DCA and MET significantly inhibited tumor growth and increased overall survival in mice.
    CONCLUSION: Compounds targeting tumor cell metabolism could become potential treatment options for glioblastoma multiforme.
    Keywords:  Dichloroacetate; Warburg effect; glioblastoma; metformin
  16. Biochim Biophys Acta Bioenerg. 2021 Jan 05. pii: S0005-2728(20)30215-2. [Epub ahead of print] 148365
      Mitochondria are highly dynamic and stress-responsive organelles that are renewed, maintained and removed by a number of different mechanisms. Recent findings bring more evidence for the focused, defined, and regulatory function of the intramitochondrial proteases extending far beyond the traditional concepts of damage control and stress responses. Until recently, the macrodegradation processes, such as mitophagy, were promoted as the major regulator of OXPHOS remodelling and turnover. However, the spatiotemporal dynamics of the OXPHOS system can be greatly modulated by the intrinsic mitochondrial mechanisms acting apart from changes in the global mitochondrial dynamics. This, in turn, may substantially contribute to the shaping of the metabolic status of the cell.
    Keywords:  Mitochondrial respiratory chain; OXPHOS maintanance; OXPHOS turnover; mitochondrial proteases
  17. Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2016778118. [Epub ahead of print]118(4):
      O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.
    Keywords:  HCF-1; O-GlcNAc transferase; OGT; cell proliferation; enzyme
  18. Biology (Basel). 2021 Jan 06. pii: E33. [Epub ahead of print]10(1):
      Cancer is one of the world's deadliest afflictions. Despite recent advances in diagnostic and surgical technologies, as well as improved treatments of some individual tumor types, there is currently no universal cure to prevent or impede the uncontrolled proliferation of malignant cells. Targeting tumors by inducing apoptosis is one of the pillars of cancer treatment. Changes in mitochondrial morphology precede intrinsic apoptosis, but mitochondrial dynamics has only recently been recognized as a viable pharmacological target. In many cancers, oncogenic transformation is accompanied by accumulation of elevated cellular levels of ROS leading to redox imbalance. Hence, a common chemotherapeutic strategy against such tumor types involves deploying pro-oxidant agents to increase ROS levels above an apoptotic death-inducing threshold. The aim of this chapter is to investigate the benefit of stimulating mitochondrial fission-dependent production of ROS for enhanced killing of solid tumors. The main question to be addressed is whether a sudden and abrupt change in mitochondrial shape toward the fragmented phenotype can be pharmacologically harnessed to trigger a burst of mitochondrial ROS sufficient to initiate apoptosis specifically in cancer cells but not in non-transformed healthy tissues.
    Keywords:  apoptosis; cancer; chemotherapy; cyclin C; mitochondria; mitochondrial dynamics; mitophagy; oxidative stress; reactive oxygen species; stress signaling
  19. Rev Physiol Biochem Pharmacol. 2021 Jan 05.
      Being originally discovered as cellular recycling bins, lysosomes are today recognized as versatile signaling organelles that control a wide range of cellular functions that are essential not only for the well-being of normal cells but also for malignant transformation and cancer progression. In addition to their core functions in waste disposal and recycling of macromolecules and energy, lysosomes serve as an indispensable support system for malignant phenotype by promoting cell growth, cytoprotective autophagy, drug resistance, pH homeostasis, invasion, metastasis, and genomic integrity. On the other hand, malignant transformation reduces the stability of lysosomal membranes rendering cancer cells sensitive to lysosome-dependent cell death. Notably, many clinically approved cationic amphiphilic drugs widely used for the treatment of other diseases accumulate in lysosomes, interfere with their cancer-promoting and cancer-supporting functions and destabilize their membranes thereby opening intriguing possibilities for cancer therapy. Here, we review the emerging evidence that supports the supplementation of current cancer therapies with lysosome-targeting cationic amphiphilic drugs.
    Keywords:  Cancer; Cathepsins; Cationic amphiphilic drugs; Cell death; Lysosome; SMPD1; pH
  20. J Med Chem. 2021 Jan 04.
      Mitochondrial dysfunction is an underlying pathology in numerous diseases. Delivery of diagnostic and therapeutic cargo directly into mitochondria is a powerful approach to study and treat these diseases. The triphenylphosphonium (TPP+) moiety is the most widely used mitochondriotropic carrier. However, studies have shown that TPP+ is not inert; TPP+ conjugates uncouple mitochondrial oxidative phosphorylation. To date, all efforts toward addressing this problem have focused on modifying lipophilicity of TPP+-linker-cargo conjugates to alter mitochondrial uptake, albeit with limited success. We show that structural modifications to the TPP+ phenyl rings that decrease electron density on the phosphorus atom can abrogate uncoupling activity as compared to the parent TPP+ moiety and prevent dissipation of mitochondrial membrane potential. These alterations of the TPP+ structure do not negatively affect the delivery of cargo to mitochondria. Results here identify the 4-CF3-phenyl TPP+ moiety as an inert mitochondria-targeting carrier to safely target pharmacophores and probes to mitochondria.
  21. Nat Commun. 2021 01 04. 12(1): 26
      Mitochondrial oxidation-induced cell death, a physiological process triggered by various cancer therapeutics to induce oxidative stress on tumours, has been challenging to investigate owing to the difficulties in generating mitochondria-specific oxidative stress and monitoring mitochondrial responses simultaneously. Accordingly, to the best of our knowledge, the relationship between mitochondrial protein oxidation via oxidative stress and the subsequent cell death-related biological phenomena has not been defined. Here, we developed a multifunctional iridium(III) photosensitiser, Ir-OA, capable of inducing substantial mitochondrial oxidative stress and monitoring the corresponding change in viscosity, polarity, and morphology. Photoactivation of Ir-OA triggers chemical modifications in mitochondrial protein-crosslinking and oxidation (i.e., oxidative phosphorylation complexes and channel and translocase proteins), leading to microenvironment changes, such as increased microviscosity and depolarisation. These changes are strongly related to cell death by inducing mitochondrial swelling with excessive fission and fusion. We suggest a potential mechanism from mitochondrial oxidative stress to cell death based on proteomic analyses and phenomenological observations.
  22. Cell Rep. 2021 Jan 05. pii: S2211-1247(20)31568-0. [Epub ahead of print]34(1): 108579
      O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.
    Keywords:  O-GlcNAcylation; O-linked N-acetylglucosamine transferase; OGT; PINK1; hematopoietic progenitor cell; hematopoietic stem cell; mitochondria; mitophagy
  23. Front Cell Dev Biol. 2020 ;8 610266
      Pseudoxanthoma elasticum (PXE) is a genetic disease considered as a paradigm of ectopic mineralization disorders, being characterized by multisystem clinical manifestations due to progressive calcification of skin, eyes, and the cardiovascular system, resembling an age-related phenotype. Although fibroblasts do not express the pathogenic ABCC6 gene, nevertheless these cells are still under investigation because they regulate connective tissue homeostasis, generating the "arena" where cells and extracellular matrix components can promote pathologic calcification and where activation of pro-osteogenic factors can be associated to pathways involving mitochondrial metabolism. The aim of the present study was to integrate structural and bioenergenetic features to deeply investigate mitochondria from control and from PXE fibroblasts cultured in standard conditions and to explore the role of mitochondria in the development of the PXE fibroblasts' pathologic phenotype. Proteomic, biochemical, and morphological data provide new evidence that in basal culture conditions (1) the protein profile of PXE mitochondria reveals a number of differentially expressed proteins, suggesting changes in redox balance, oxidative phosphorylation, and calcium homeostasis in addition to modified structure and organization, (2) measure of oxygen consumption indicates that the PXE mitochondria have a low ability to cope with a sudden increased need for ATP via oxidative phosphorylation, (3) mitochondrial membranes are highly polarized in PXE fibroblasts, and this condition contributes to increased reactive oxygen species levels, (4) ultrastructural alterations in PXE mitochondria are associated with functional changes, and (5) PXE fibroblasts exhibit a more abundant, branched, and interconnected mitochondrial network compared to control cells, indicating that fusion prevail over fission events. In summary, the present study demonstrates that mitochondria are modified in PXE fibroblasts. Since mitochondria are key players in the development of the aging process, fibroblasts cultured from aged individuals or aged in vitro are more prone to calcify, and in PXE, calcified tissues remind features of premature aging syndromes; it can be hypothesized that mitochondria represent a common link contributing to the development of ectopic calcification in aging and in diseases. Therefore, ameliorating mitochondrial functions and cell metabolism could open new strategies to positively regulate a number of signaling pathways associated to pathologic calcification.
    Keywords:  OCR; PXE; fibroblast; mitochondria; morphology; proteome; ultrastructure
  24. Oncogene. 2021 Jan 08.
      The advanced or recurrent endometrial cancer (EC) has a poor prognosis because of chemoresistance. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is overexpressed in a variety of human cancers and plays important roles in promoting tumor cell growth. Here, we showed that high expression of PFKFB3 in EC cell lines is associated with chemoresistance. Pharmacological inhibition of PFKFB3 with PFK158 and or genetic downregulation of PFKFB3 dramatically suppressed cell proliferation and enhanced the sensitivity of EC cells to carboplatin (CBPt) and cisplatin (Cis). Moreover, PFKFB3 inhibition resulted in reduced glucose uptake, ATP production, and lactate release. Notably, we found that PFK158 with CBPt or Cis exerted strong synergistic antitumor activity in chemoresistant EC cell lines, HEC-1B and ARK-2 cells. We also found that the combination of PFK158 and CBPt/Cis induced apoptosis- and autophagy-mediated cell death through inhibition of the Akt/mTOR signaling pathway. Mechanistically, we found that PFK158 downregulated the CBPt/Cis-induced upregulation of RAD51 expression and enhanced CBPt/Cis-induced DNA damage as demonstrated by an increase in γ-H2AX levels in HEC-1B and ARK-2 cells, potentially revealing a means to enhance PFK158-induced chemosensitivity. More importantly, PFK158 treatment, either as monotherapy or in combination with CBPt, led to a marked reduction in tumor growth in two chemoresistant EC mouse xenograft models. These data suggest that PFKFB3 inhibition alone or in combination with standard chemotherapy may be used as a novel therapeutic strategy for improved therapeutic efficacy and outcomes of advanced and recurrent EC patients.
  25. Cancer Metab. 2021 Jan 07. 9(1): 1
      BACKGROUND: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated.METHODS: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo.
    RESULTS: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo.
    CONCLUSIONS: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.
    Keywords:  AAR; ATF4; Activating transcription factor 4; Amino acid deprivation; Amino acid response; Cancer metabolism; Cataplerosis; ER stress; GCN2; PCK2; PEP; PEPCK; PEPCK-M; PKC-ζ; PRODH/POX; PYCR; Phosphoenolpyruvate; Phosphoenolpyruvate carboxykinase; Proline metabolism; Serine/glycine metabolism; TCA cycle
  26. Mitochondrion. 2021 Jan 04. pii: S1567-7249(20)30243-9. [Epub ahead of print]
      C-Src kinase is localized in several subcellular compartments, including mitochondria where it is involved in the regulation of organelle functions and overall metabolism. Surprisingly, the characterization of the intramitochondrial Src interactome has never been fully determined. Using in vitro proximity-dependent biotin identification (BioID) coupled to mass spectrometry, we identified 51 candidate proteins that may interact directly or indirectly with c-Src within the mitochondrial matrix. Pathway analysis suggests that these proteins are involved in a large array of mitochondrial functions such as protein folding and import, mitochondrial organization and transport, oxidative phosphorylation, tricarboxylic acid cycle and metabolism of amino and fatty acids. Among these proteins, we identified 24 tyrosine phosphorylation sites in 17 mitochondrial proteins (AKAP1, VDAC1, VDAC2, VDAC3, LonP1, Hsp90, SLP2, PHB2, MIC60, UBA1, EF-Tu, LRPPRC, ACO2, OAT, ACAT1, ETFβ and ATP5β) as potential substrates for intramitochondrial Src using in silico prediction of tyrosine phospho-sites. Interaction of c-Src with SLP2 and ATP5β was confirmed using coimmunoprecipitation. This study suggests that the intramitochondrial Src could target several proteins and regulate different mitochondrial functions.
    Keywords:  BioID2; Mitochondrial functions; c-Src kinase; protein-protein interactions; proteomics
  27. Elife. 2021 Jan 08. pii: e60191. [Epub ahead of print]10
      The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.
    Keywords:  MYC; cancer; cancer biology; human; mouse; ribosome biogenesis
  28. Nat Genet. 2021 Jan;53(1): 86-99
      Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
  29. Sci Transl Med. 2021 Jan 06. pii: eaba6110. [Epub ahead of print]13(575):
      Although chemotherapeutic agents have been used for decades, the mechanisms of action, mechanisms of resistance, and the best treatment schedule remain elusive. Mitomycin C (MMC) is the gold standard treatment for non-muscle-invasive bladder cancer (NMIBC). However, it is effective only in a subset of patients, suggesting that, aside from cytotoxicity, other mechanisms could be involved in mediating the success of the treatment. Here, we showed that MMC promotes immunogenic cell death (ICD) and in vivo tumor protection. MMC-induced ICD relied on metabolic reprogramming of tumor cells toward increased oxidative phosphorylation. This favored increased mitochondrial permeability leading to the cytoplasmic release of mitochondrial DNA, which activated the inflammasome for efficient IL-1β (interleukin-1β) secretion that promoted dendritic cell maturation. Resistance to ICD was associated with mitochondrial dysfunction related to low abundance of complex I of the respiratory chain. Analysis of complex I in patient tumors indicated that low abundance of this mitochondrial complex was associated with recurrence incidence after chemotherapy in patients with NMIBC. The identification of mitochondria-mediated ICD as a mechanism of action of MMC offers opportunities to optimize bladder cancer management and provides potential markers of treatment efficacy that could be used for patient stratification.
  30. Front Oncol. 2020 ;10 570656
      Overexpression of DGUOK promotes mitochondria oxidative phosphorylation and lung adenocarcinoma progression. However, the role and mechanism of DGUOK in regulation of mitochondria function and lung cancer progression still poorly understood. Here we demonstrated that DGUOK regulated NAD+ biogenesis. Depletion of the DGUOK significantly decreased NAD+ level. Furthermore, knockout of the DGUOK considerably reduced expression of the NMNAT2, a key molecule controlling NAD+ synthesis, at both mRNA and protein levels. Ectopic expression of the NMNAT2 abrogated the effect of knockdown of DGUOK on NAD+. Notably, this regulation is independent of DGUOK -mediated mitochondria complex I activity. We also showed that NMNAT2 was highly expressed in lung adenocarcinoma and negatively correlated with the patient overall survival. Our study suggested that DGUOK regulates NAD+ in a NMNAT2 dependent manner and DGUOK-NMNAT2-NAD+ axis could be a potential therapeutic target in lung adenocarcinoma.
    Keywords:  NAD+; NMNAT2; deoxyguanosine kinase; lung adenocarcinoma; mitochondria complex I
  31. Mol Metab. 2021 Jan 02. pii: S2212-8778(20)30234-9. [Epub ahead of print] 101160
      OBJECTIVE: The immediate signals that couple exercise to metabolic adaptation are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a producer of reactive oxygen species (ROS) and plays a significant role in both metabolic and vascular adaptation during conditions of stress. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism.METHODS: Mice were subjected to acute exercise to assess immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and subsequent substrate oxidation was measured.
    RESULTS: We identified key exercise-responsive metabolic genes that are dependent on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Furthermore, Nox4-deficient mice had decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Nox4-deficient mice also demonstrated impaired adaptation to chronic exercise as measured by time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise.
    CONCLUSION: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting that the endothelium is important for the skeletal muscle metabolic response to exercise.
    Keywords:  Acute exercise; Nox4; ROS; exercise training; hydrogen peroxide; metabolic adaptation; skeletal muscle metabolism
  32. Cell Metab. 2021 Jan 05. pii: S1550-4131(20)30666-5. [Epub ahead of print]33(1): 78-93.e7
      Obesity is often linked to malignancies including multiple myeloma, and the underlying mechanisms remain elusive. Here we showed that acetyl-CoA synthetase 2 (ACSS2) may be an important linker in obesity-related myeloma. ACSS2 is overexpressed in myeloma cells derived from obese patients and contributes to myeloma progression. We identified adipocyte-secreted angiotensin II as a direct cause of adiposity in increased ACSS2 expression. ACSS2 interacts with oncoprotein interferon regulatory factor 4 (IRF4), and enhances IRF4 stability and IRF4-mediated gene transcription through activation of acetylation. The importance of ACSS2 overexpression in myeloma is confirmed by the finding that an inhibitor of ACSS2 reduces myeloma growth both in vitro and in a diet-induced obese mouse model. Our findings demonstrate a key impact for obesity-induced ACSS2 on the progression of myeloma. Given the central role of ACSS2 in many tumors, this mechanism could be important to other obesity-related malignancies.
    Keywords:  ACSS2; IRF4; adipocytes; angiotensin II; autophagy; lysine acetylation; multiple myeloma; obesity
  33. Nat Commun. 2021 01 04. 12(1): 83
      Trafficking of mitochondria into dendrites and axons plays an important role in the physiology and pathophysiology of neurons. Mitochondrial outer membrane protein Miro and adaptor proteins TRAKs/Milton link mitochondria to molecular motors. Here we show that metaxins MTX-1 and MTX-2 contribute to mitochondrial transport into both dendrites and axons of C. elegans neurons. MTX1/2 bind to MIRO-1 and kinesin light chain KLC-1, forming a complex to mediate kinesin-1-based movement of mitochondria, in which MTX-1/2 are essential and MIRO-1 plays an accessory role. We find that MTX-2, MIRO-1, and TRAK-1 form another distinct adaptor complex to mediate dynein-based transport. Additionally, we show that failure of mitochondrial trafficking in dendrites causes age-dependent dendrite degeneration. We propose that MTX-2 and MIRO-1 form the adaptor core for both motors, while MTX-1 and TRAK-1 specify each complex for kinesin-1 and dynein, respectively. MTX-1 and MTX-2 are also required for mitochondrial transport in human neurons, indicative of their evolutionarily conserved function.
  34. Nat Metab. 2021 Jan 04.
      Metabolic transformation is a hallmark of cancer and a critical target for cancer therapy. Cancer metabolism and behaviour are regulated by cell-intrinsic factors as well as metabolite availability in the tumour microenvironment (TME). This metabolic niche within the TME is shaped by four tiers of regulation: (1) intrinsic tumour cell metabolism, (2) interactions between cancer cells and non-cancerous cells, (3) tumour location and heterogeneity and (4) whole-body metabolic homeostasis. Here, we define these modes of metabolic regulation and review how distinct cell types contribute to the metabolite composition of the TME. Finally, we connect these insights to understand how each of these tiers offers unique therapeutic potential to modulate the metabolic profile and function of all cells inhabiting the TME.
  35. Nat Commun. 2021 01 04. 12(1): 102
      Pro-inflammatory activation of adipose tissue macrophages (ATMs) is causally linked to obesity and obesity-associated disorders. A number of studies have demonstrated the crucial role of mitochondrial metabolism in macrophage activation. However, there is a lack of pharmaceutical agents to target the mitochondrial metabolism of ATMs for the treatment of obesity-related diseases. Here, we characterize a near-infrared fluorophore (IR-61) that preferentially accumulates in the mitochondria of ATMs and has a therapeutic effect on diet-induced obesity as well as obesity-associated insulin resistance and fatty liver. IR-61 inhibits the classical activation of ATMs by increasing mitochondrial complex levels and oxidative phosphorylation via the ROS/Akt/Acly pathway. Taken together, our findings indicate that specific enhancement of ATMs oxidative phosphorylation improves chronic inflammation and obesity-related disorders. IR-61 might be an anti-inflammatory agent useful for the treatment of obesity-related diseases by targeting the mitochondria of ATMs.
  36. Redox Biol. 2020 Dec 28. pii: S2213-2317(20)31057-0. [Epub ahead of print]40 101852
      In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.
    Keywords:  EPR; ESR; ETC; Hydroxylamine; Mitochondria; Nitroxide; Oxygen consumption rate (OCR); Superoxide
  37. J Cancer. 2021 ;12(1): 232-243
      Background and aim: Silencing the expression of ACACA inhibits cell proliferation and induces apoptosis in prostate cancer LNCaP cells. However, the role of ACACA in other prostate cancer cells is not fully understood. Also, the effect of knocking down ACACA gene on mitochondria remains unclear. This study aimed to discover the specific role of ACACA gene in prostate cancer (PCa) DU145 and PC3 cells as well as its effects on mitochondrial potential. Methods: The expression of ACACA gene was detected in human prostate cancer tissue microarrays and assessed in different clinical stages. Then, prostate cancer cell lines with low expression of ACACA were constructed to evaluate the changes in their cell cycle, proliferation, and metabolites. The effect of ACACA on tumor formation in vivo was analyzed. Also, mito-ATP production, mitochondrial staining, and mtDNA, nicotinamide adenine dinucleotide (NAD+/NADH), and reactive oxygen species (ROS) levels were detected. Results: ACACA was expressed more strongly in prostate cancer tissues. The expression level of ACACA was higher in patients with advanced PCa than in patients with lower grades. The proliferation ability reduced in ACACA-knockdown cells. In in vivo tests, the tumor volume and weight were lower in the experimental group than in the control group. Mito-ATP production decreased significantly after ACACA suppression, mtDNA levels and MitoTracker staining decreased in the experimental group. The ratio of NAD+/NADH and ROS levels were upregulated in the experimental group. Conclusion: Targeting ACACA gene and mitochondria might serve as a novel therapy for prostate cancer treatment.
    Keywords:  ACACA; DU145; Mitochondria; PC3; Prostate cancer
  38. Cell Rep. 2021 Jan 05. pii: S2211-1247(20)31572-2. [Epub ahead of print]34(1): 108583
      Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. Here, we report a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction.
    Keywords:  Drosophila; metabolomics; microbiome; mitochondria; oogenesis; riboflavin
  39. Cell. 2021 Jan 07. pii: S0092-8674(20)31535-X. [Epub ahead of print]184(1): 226-242.e21
      Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.
    Keywords:  MRD; autophagy; barcode; chemotherapy; colorectal cancer; diapause; drug tolerant persisters; equipotent; mTOR; slow-cycling
  40. Sci Signal. 2021 Jan 05. pii: eabc4436. [Epub ahead of print]14(664):
      Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.
  41. Nat Immunol. 2021 Jan 04.
      Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.
  42. Nat Med. 2021 Jan 04.
      Metastasis is the primary cause of cancer mortality, and cancer frequently metastasizes to the liver. It is not clear whether liver immune tolerance mechanisms contribute to cancer outcomes. We report that liver metastases diminish immunotherapy efficacy systemically in patients and preclinical models. Patients with liver metastases derive limited benefit from immunotherapy independent of other established biomarkers of response. In multiple mouse models, we show that liver metastases siphon activated CD8+ T cells from systemic circulation. Within the liver, activated antigen-specific Fas+CD8+ T cells undergo apoptosis following their interaction with FasL+CD11b+F4/80+ monocyte-derived macrophages. Consequently, liver metastases create a systemic immune desert in preclinical models. Similarly, patients with liver metastases have reduced peripheral T cell numbers and diminished tumoral T cell diversity and function. In preclinical models, liver-directed radiotherapy eliminates immunosuppressive hepatic macrophages, increases hepatic T cell survival and reduces hepatic siphoning of T cells. Thus, liver metastases co-opt host peripheral tolerance mechanisms to cause acquired immunotherapy resistance through CD8+ T cell deletion, and the combination of liver-directed radiotherapy and immunotherapy could promote systemic antitumor immunity.
  43. Nat Commun. 2021 01 04. 12(1): 98
      Glucose metabolism and innate immunity evolved side-by-side. It is unclear if and how the two systems interact with each other during hepatitis B virus (HBV) infections and, if so, which mechanisms are involved. Here, we report that HBV activates glycolysis to impede retinoic acid-inducible gene I (RIG-I)-induced interferon production. We demonstrate that HBV sequesters MAVS from RIG-I by forming a ternary complex including hexokinase (HK). Using a series of pharmacological and genetic approaches, we provide in vitro and in vivo evidence indicating that HBV suppresses RLR signaling via lactate dehydrogenase-A-dependent lactate production. Lactate directly binds MAVS preventing its aggregation and mitochondrial localization during HBV infection. Therefore, we show that HK2 and glycolysis-derived lactate have important functions in the immune escape of HBV and that energy metabolism regulates innate immunity during HBV infection.
  44. J Cancer. 2021 ;12(3): 630-643
      Ovarian cancer (OC) is the most lethal of gynecological tumors in women. Tumor metabolism has become a new opportunity in the treatment of tumors. Pyruvate dehydrogenase kinase 1 (PDK1), as a key regulatory enzyme implicated in metabolic reprogramming of tumors, abnormally high expressed in various tumors and involved in the regulation of tumor cell biological behavior. However, the role of PDK1 in the occurrence and development of ovarian cancer remains unclear. Our team identified the expression of PDK1 in ovarian cancer cell lines and tissues through RT-PCR and immunohistochemical staining and evaluated the correlation of PDK1 expression with clinicopathologic features of patients and survival analyses. We used a variety of in vitro experiments to explore the influence of PDK1 on proliferation, invasion, migration, colony formation, apoptosis and the cell cycle of ovarian cancer cell lines CAOV3 and SKOV3. PDK1 was highly expressed in ovarian cancer cell lines and OC tissues. High expression of PDK1 was closely correlated to tumor size, FIGO stage, extraovarian metastases status and distribution. Univariate and multivariate Cox regression analysis identified that PDK1 was an independent prognostic factor for overall survival. Moreover, PDK1 was a superior predictor in prognosis of ovarian cancer and the incorporation of CA125 into PDK1 generated a predictive combination that displayed better predictive accuracy for overall survival. Downregulation of PDK1 suppressed the biological behavior of ovarian cancer cells due to S phase arrest and cellular apoptosis. PDK1 may serve as a novel prognostic biomarker, even a promising antineoplastic target of ovarian cancer.
    Keywords:  Pyruvate dehydrogenase kinase 1; biological behavior; ovarian cancer; prognostic biomarker; tumor metabolic
  45. Theranostics. 2021 ;11(4): 1828-1844
      This study aimed to screen novel anticancer strategies from FDA-approved non-cancer drugs and identify potential biomarkers and therapeutic targets for colorectal cancer (CRC). Methods: A library consisting of 1056 FDA-approved drugs was screened for anticancer agents. WST-1, colony-formation, flow cytometry, and tumor xenograft assays were used to determine the anticancer effect of azelastine. Quantitative proteomics, confocal imaging, Western blotting and JC-1 assays were performed to examine the effects on mitochondrial pathways. The target protein of azelastine was analyzed and confirmed by DARTS, WST-1, Biacore and tumor xenograft assays. Immunohistochemistry, gain- and loss-of-function experiments, WST-1, colony-formation, immunoprecipitation, and tumor xenograft assays were used to examine the functional and clinical significance of ARF1 in colon tumorigenesis. Results: Azelastine, a current anti-allergic drug, was found to exert a significant inhibitory effect on CRC cell proliferation in vitro and in vivo, but not on ARF1-deficient or ARF1-T48S mutant cells. ARF1 was identified as a direct target of azelastine. High ARF1 expression was associated with advanced stages and poor survival of CRC. ARF1 promoted colon tumorigenesis through its interaction with IQGAP1 and subsequent activation of ERK signaling and mitochondrial fission by enhancing the interaction of IQGAP1 with MEK and ERK. Mechanistically, azelastine bound to Thr-48 in ARF1 and repressed its activity, decreasing Drp1 phosphorylation. This, in turn, inhibited mitochondrial fission and suppressed colon tumorigenesis by blocking IQGAP1-ERK signaling. Conclusions: This study provides the first evidence that azelastine may be novel therapeutics for CRC treatment. ARF1 promotes colon tumorigenesis, representing a promising biomarker and therapeutic target in CRC.
    Keywords:  ARF1; azelastine; colorectal cancer; drug repurposing; mitochondrial fission
  46. Nat Genet. 2021 Jan;53(1): 11-15
      In multiple cancer types, high tumor mutational burden (TMB) is associated with longer survival after treatment with immune checkpoint inhibitors (ICIs). The association of TMB with survival outside of the immunotherapy context is poorly understood. We analyzed 10,233 patients (80% non-ICI-treated, 20% ICI-treated) with 17 cancer types before/without ICI treatment or after ICI treatment. In non-ICI-treated patients, higher TMB (higher percentile within cancer type) was not associated with better prognosis; in fact, in many cancer types, higher TMB was associated with poorer survival, in contrast to ICI-treated patients in whom higher TMB was associated with longer survival.
  47. Cell Death Dis. 2021 Jan 07. 12(1): 26
      Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.
  48. Oncotarget. 2020 Dec 15. 11(50): 4613-4624
      Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells.
    Keywords:  OXPHOS; energy source; glycolysis; metabolism; polyphosphate
  49. EMBO Rep. 2021 Jan 04. e51162
      Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity.
    Keywords:  OTUD1; colorectal cancer; ferroptosis; iron transportation
  50. Cell Metab. 2021 Jan 05. pii: S1550-4131(20)30665-3. [Epub ahead of print]33(1): 160-173.e6
      CD147 is a tumor-associated glycoprotein that regulates cell metabolism. However, CD147 methylation and its subsequent role in cancer cell metabolism remain unclear. Here, we detect CD147 di-methylation in 16 non-small-cell lung cancer (NSCLC) tissues using liquid chromatography-tandem mass spectrometry. CD147 is di-methylated to CD147-K234me2 by lysine methyltransferase 5A (KMT5A). The increase in KMT5A expression boosts the levels of CD147-K234me2, further promoting the interaction between CD147 and monocarboxylate transporter 4 (MCT4), which enhances the translocation of MCT4 from the cytoplasm to the membrane. Overexpression of CD147-K234me2 and KMT5A enhances glycolysis and lactate export in NSCLC cells. Clinical analysis shows that high CD147-K234me2 expression is significantly related to cancer progression and overall survival, and has prognostic significance in individuals with NSCLC, especially for those in the early stages. Our findings indicate that CD147-K234me2 plays a critical role in cancer metabolism, and it can be a highly promising therapeutic target for NSCLC.
    Keywords:  CD147; KMT5A; MCT4; NSCLC; di-methylation; lactate