bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒05‒02
forty papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Cell Mol Life Sci. 2021 Apr 24.
      Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.
    Keywords:  Glutaminase; Glutamine metabolism; Mitochondrial fission; ROS; Tumor growth
  2. J Clin Invest. 2021 Apr 29. pii: 143691. [Epub ahead of print]
      Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factor hypoxia-inducible factor (HIF)-1α and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult and tumors rapidly acquire resistance to recently developed inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α expressing tumor enteroids. Our work demonstrates that HIF-2α integrates two independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upreguated lipid and iron regulatory genes in colon cancer cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Secondly, via an iron dependent, lipid peroxidation-independent pathway, HIF-2α activation potentiated ROS, via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrate a mechanistic vulnerability in cancer cells that were the dependent on HIF-2α that can be leveraged for colon cancer treatment.
    Keywords:  Cancer; Cell stress; Hypoxia; Metabolism; Oncology
  3. Molecules. 2021 Apr 15. pii: 2303. [Epub ahead of print]26(8):
      Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.
    Keywords:  MCL-1 inhibitor S63845; acute myeloid leukemia (AML); metformin; reactive oxygen species (ROS)
  4. Cancers (Basel). 2021 Apr 06. pii: 1727. [Epub ahead of print]13(7):
      Tumor cells display metabolic alterations when compared to non-transformed cells. These characteristics are crucial for tumor development, maintenance and survival providing energy supplies and molecular precursors. Anaplerosis is the property of replenishing the TCA cycle, the hub of carbon metabolism, participating in the biosynthesis of precursors for building blocks or signaling molecules. In advanced prostate cancer, an upshift of succinate-driven oxidative phosphorylation via mitochondrial Complex II was reported. Here, using untargeted metabolomics, we found succinate accumulation mainly in malignant cells and an anaplerotic effect contributing to biosynthesis, amino acid, and carbon metabolism. Succinate also stimulated oxygen consumption. Malignant prostate cells displayed higher mitochondrial affinity for succinate when compared to non-malignant prostate cells and the succinate-driven accumulation of metabolites induced expression of mitochondrial complex subunits and their activities. Moreover, extracellular succinate stimulated migration, invasion, and colony formation. Several enzymes linked to accumulated metabolites in the malignant cells were found upregulated in tumor tissue datasets, particularly NME1 and SHMT2 mRNA expression. High expression of the two genes was associated with shorter disease-free survival in prostate cancer cohorts. Moreover, in-vitro expression of both genes was enhanced in prostate cancer cells upon succinate stimulation. In conclusion, the data indicate that uptake of succinate from the tumor environment has an anaplerotic effect that enhances the malignant potential of prostate cancer cells.
    Keywords:  anaplerosis; cancer metabolism; mitochondria; prostate cancer; succinate
  5. Antioxidants (Basel). 2021 Apr 15. pii: 609. [Epub ahead of print]10(4):
      Mitochondria are popularly called the "powerhouses" of the cell. They promote energy metabolism through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, which in contrast to cytosolic glycolysis are oxygen-dependent and significantly more substrate efficient. That is, mitochondrial metabolism provides substantially more cellular energy currency (ATP) per macronutrient metabolised. Enhancement of mitochondrial density and metabolism are associated with endurance training, which allows for the attainment of high relative VO2 max values. However, the sedentary lifestyle and diet currently predominant in the Western world lead to mitochondrial dysfunction. Underdeveloped mitochondrial metabolism leads to nutrient-induced reducing pressure caused by energy surplus, as reduced nicotinamide adenine dinucleotide (NADH)-mediated high electron flow at rest leads to "electron leak" and a chronic generation of superoxide radicals (O2-). Chronic overload of these reactive oxygen species (ROS) damages cell components such as DNA, cell membranes, and proteins. Counterintuitively, transiently generated ROS during exercise contributes to adaptive reduction-oxidation (REDOX) signalling through the process of cellular hormesis or "oxidative eustress" defined by Helmut Sies. However, the unaccustomed, chronic oxidative stress is central to the leading causes of mortality in the 21st century-metabolic syndrome and the associated cardiovascular comorbidities. The endurance exercise training that improves mitochondrial capacity and the protective antioxidant cellular system emerges as a universal intervention for mitochondrial dysfunction and resultant comorbidities. Furthermore, exercise might also be a solution to prevent ageing-related degenerative diseases, which are caused by impaired mitochondrial recycling. This review aims to break down the metabolic components of exercise and how they translate to athletic versus metabolically diseased phenotypes. We outline a reciprocal relationship between oxidative metabolism and inflammation, as well as hypoxia. We highlight the importance of oxidative stress for metabolic and antioxidant adaptation. We discuss the relevance of lactate as an indicator of critical exercise intensity, and inferring from its relationship with hypoxia, we suggest the most appropriate mode of exercise for the case of a lost oxidative identity in metabolically inflexible patients. Finally, we propose a reciprocal signalling model that establishes a healthy balance between the glycolytic/proliferative and oxidative/prolonged-ageing phenotypes. This model is malleable to adaptation with oxidative stress in exercise but is also susceptible to maladaptation associated with chronic oxidative stress in disease. Furthermore, mutations of components involved in the transcriptional regulatory mechanisms of mitochondrial metabolism may lead to the development of a cancerous phenotype, which progressively presents as one of the main causes of death, alongside the metabolic syndrome.
    Keywords:  endurance exercise; metabolic disease; oxidative phenotype; oxidative stress
  6. Antioxidants (Basel). 2021 Apr 14. pii: 603. [Epub ahead of print]10(4):
      Metabolic reprogramming, which is characteristic of cancer cells that rapidly adapt to the hypoxic microenvironment and is crucial for tumor growth and metastasis, is recognized as one of the major mechanisms underlying therapeutic resistance. Mitochondria, which are directly involved in metabolic reprogramming, are used to design novel mitochondria-targeted anticancer agents. Despite being targeted by melatonin, the functional role of mitochondria in melatonin's oncostatic activity remains unclear. In this study, we aim to investigate the role of melatonin in mitochondrial metabolism and its functional consequences in head and neck cancer. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 100, 500, and 1500 µM of melatonin for 1, 3, and 5 days, and found a connection between a change of metabolism following melatonin treatment and its effects on mitochondria. Our results demonstrate that melatonin induces a shift to an aerobic mitochondrial metabolism that is associated with changes in mitochondrial morphology, function, fusion, and fission in HNSCC. We found that melatonin increases oxidative phosphorylation (OXPHOS) and inhibits glycolysis in HNSCC, resulting in increased ROS production, apoptosis, and mitophagy, and decreased cell proliferation. Our findings highlight new molecular pathways involved in melatonin's oncostatic activity, suggesting that it could act as an adjuvant agent in a potential therapy for cancer patients. We also found that high doses of melatonin, such as those used in this study for its cytotoxic impact on HNSCC cells, might lead to additional effects through melatonin receptors.
    Keywords:  OXPHOS; apoptosis; free radicals; glycolysis; head and neck cancer cells; melatonin; mitochondria; mitophagy
  7. Cancer Metab. 2021 Apr 29. 9(1): 19
      BACKGROUND: Tumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR -ve breast cancer cells is not well understood.METHODS: In the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients.
    RESULTS: The proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR -ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients.
    CONCLUSION: The evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR -ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.
    Keywords:  Breast cancer heterogeneity; Inflammation; Metabolism; Mitochondria; TNF-α
  8. Aging (Albany NY). 2021 Apr 26. 13
      Hepatocellular carcinoma is a common type of liver cancer. Resistance to chemotherapeutic agents is a major problem in cancer therapy. MicroRNAs have been reported in cancer development and tumor growth; however, the relationship between chemoresistance and hepatocellular carcinoma needs to be fully investigated. Here, we treated hepatocellular carcinoma cell line (HA22T) with a histone deacetylase inhibitor to establish hepatocellular carcinoma-resistant cells (HDACi-R) and investigated the molecular mechanisms of chemoresistance in HCC cells. Although histone deacetylase inhibitor could not enhance cell death in HDACi-R but upregulation of miR-107 decreased cell viability both in parental cells and resistance cells, decreased the expression of cofilin-1, enhanced ROS-induced cell apoptosis, and dose-dependently sensitized HDACi-R to HDACi. Further, miR-107 upregulation resulted in tumor cell disorganization in both HA22T and HDACi-R in a mice xenograft model. Our findings demonstrated that miR-107 downregulation leads to hepatocellular carcinoma cell resistance in HDACi via a cofilin-1-dependent molecular mechanism and ROS accumulation.
    Keywords:  chemosensitivity; drug resistance; hepatocellular carcinoma; miR-107
  9. Cancers (Basel). 2021 Apr 04. pii: 1709. [Epub ahead of print]13(7):
      Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.
    Keywords:  2HG; IDH2; breast carcinoma
  10. Life (Basel). 2021 Apr 17. pii: 351. [Epub ahead of print]11(4):
      The mitochondrial respiratory chain encompasses four oligomeric enzymatic complexes (complex I, II, III and IV) which, together with the redox carrier ubiquinone and cytochrome c, catalyze electron transport coupled to proton extrusion from the inner membrane. The protonmotive force is utilized by complex V for ATP synthesis in the process of oxidative phosphorylation. Respiratory complexes are known to coexist in the membrane as single functional entities and as supramolecular aggregates or supercomplexes (SCs). Understanding the assembly features of SCs has relevant biomedical implications because defects in a single protein can derange the overall SC organization and compromise the energetic function, causing severe mitochondrial disorders. Here we describe in detail the main types of SCs, all characterized by the presence of complex III. We show that the genetic alterations that hinder the assembly of Complex III, not just the activity, cause a rearrangement of the architecture of the SC that can help to preserve a minimal energetic function. Finally, the major metabolic disturbances associated with severe SCs perturbation due to defective complex III are discussed along with interventions that may circumvent these deficiencies.
    Keywords:  MTCYB mutations; complex III; cytochrome b; mitochondrial DNA; mitochondrial diseases; oxidative stress; respiratory complexes; respiratory supercomplexes
  11. Cancers (Basel). 2021 Apr 10. pii: 1809. [Epub ahead of print]13(8):
      β-arrestin 1 (ARRB1) is a scaffold protein that regulates signaling downstream of G protein-coupled receptors (GPCRs). In the current work, we investigated the role of ARRB1 in regulating the metabolic preference of cancer stem cell (CSC)-like cells in bladder cancer (BC). We show that ARRB1 is crucial for spheroid formation and tumorigenic potential. Furthermore, we measured mitochondrial respiration, glucose uptake, glycolytic rate, mitochondrial/glycolytic ATP production and fuel oxidation in previously established ARRB1 knock out (KO) cells and corresponding controls. Our results demonstrate that depletion of ARRB1 decreased glycolytic rate and induced metabolic reprogramming towards oxidative phosphorylation. Mechanistically, the depletion of ARRB1 dramatically increased the mitochondrial pyruvate carrier MPC1 protein levels and reduced the glucose transporter GLUT1 protein levels along with glucose uptake. Overexpression of ARRB1 in ARRB1 KO cells reversed the phenotype and resulted in the upregulation of glycolysis. In conclusion, we show that ARRB1 regulates the metabolic preference of BC CSC-like cells and functions as a molecular switch that promotes reprogramming towards glycolysis by negatively regulating MPC1 and positively regulating GLUT1/ glucose uptake. These observations open new therapeutic avenues for targeting the metabolic preferences of cancer stem cell (CSC)-like BC cells.
    Keywords:  bladder cancer; cancer system cells; glucose transporter; metabolic reprograming; mitochondrial pyruvate carrier; β-arrestin 1
  12. Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00338-7. [Epub ahead of print]35(4): 109024
      Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.
    Keywords:  OXPHOS; cryo-EM; dalfopristin; drug repurposing; glioblastoma; glioblastoma stem cells; high-content screening; mitochondrial translation; mitoribosome; quinupristin
  13. Nat Metab. 2021 Apr 26.
      Cytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP-AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS-STING-TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS-STING-TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.
  14. Biomolecules. 2021 Apr 10. pii: 558. [Epub ahead of print]11(4):
      Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.
    Keywords:  electron transport chain; energetic metabolism; hematopoietic progenitors; hematopoietic stem cells; oxidative phosphorylation; proliferative capacity; quiescence; α-tocopherol acetate
  15. Biochem Soc Trans. 2021 Apr 30. pii: BST20200742. [Epub ahead of print]
      In the last two decades, IF1, the endogenous inhibitor of the mitochondrial F1Fo-ATPase (ATP synthase) has assumed greater and ever greater interest since it has been found to be overexpressed in many cancers. At present, several findings indicate that IF1 is capable of playing a central role in cancer cells by promoting metabolic reprogramming, proliferation and resistance to cell death. However, the mechanism(s) at the basis of this pro-oncogenic action of IF1 remains elusive. Here, we recall the main features of the mechanism of the action of IF1 when the ATP synthase works in reverse, and discuss the experimental evidence that support its relevance in cancer cells. In particular, a clear pro-oncogenic action of IF1 is to avoid wasting of ATP when cancer cells are exposed to anoxia or near anoxia conditions, therefore favoring cell survival and tumor growth. However, more recently, various papers have described IF1 as an inhibitor of the ATP synthase when it is working physiologically (i.e. synthethizing ATP), and therefore reprogramming cell metabolism to aerobic glycolysis. In contrast, other studies excluded IF1 as an inhibitor of ATP synthase under normoxia, providing the basis for a hot debate. This review focuses on the role of IF1 as a modulator of the ATP synthase in normoxic cancer cells with the awareness that the knowledge of the molecular action of IF1 on the ATP synthase is crucial in unravelling the molecular mechanism(s) responsible for the pro-oncogenic role of IF1 in cancer and in developing related anticancer strategies.
    Keywords:  ATP synthase; ROS; inhibitor factor 1
  16. Aging (Albany NY). 2021 Apr 30. 13
      Breast cancer is the most common malignancy in women all around the world, especially in many countries in Asia. However, antitumor drugs with unique curative effects and low toxic side-effects have not been found yet. Warangalone is an isoflavone extracted from the Cudrania tricuspidata fruit, and is reported to possess anti-inflammatory and anti-cancer activity. The purpose of this study was to determine the effects of warangalone on breast cancer cells. In this study, we found that warangalone decreased the viability of breast cancer cells by increasing the generation of reactive oxygen species (ROS) resulting in mitochondrial damage and decreased mitochondrial membrane potential (MMP). Warangalone induced mitochondrial apoptosis by increasing the BAX/BCL-2 ratio. Warangalone activated mitophagy via upregulation of PINK1 and Parkin expression and co-localization. The combination of warangalone and autophagy inhibitors or PINK1 siRNA increased the degree of cell apoptosis compared to treatment with warangalone alone. Warangalone damages mitochondria via ROS, thereby triggering PINK1/Parkin-mediated mitophagy and inducing mitochondrial apoptosis. However, autophagy/mitophagy protects against warangalone-induced mitochondrial apoptosis. A combination of warangalone and autophagy/mitophagy inhibitors may be a potential treatment for breast cancer.
    Keywords:  PINK1/Parkin; apoptosis; breast cancer; mitophagy; warangalone
  17. Nat Commun. 2021 Apr 30. 12(1): 2482
      While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.
  18. Int J Mol Sci. 2021 Apr 17. pii: 4164. [Epub ahead of print]22(8):
      The mitochondrial adenine nucleotide translocator (ANT) plays the fundamental role of gatekeeper of cellular energy flow, carrying out the reversible exchange of ADP for ATP across the inner mitochondrial membrane. ADP enters the mitochondria where, through the oxidative phosphorylation process, it is the substrate of Fo-F1 ATP synthase, producing ATP that is dispatched from the mitochondrion to the cytoplasm of the host cell, where it can be used as energy currency for the metabolic needs of the cell that require energy. Long ago, we performed a method that allowed us to monitor the activity of ANT by continuously detecting the ATP gradually produced inside the mitochondria and exported in the extramitochondrial phase in exchange with externally added ADP, under conditions quite close to a physiological state, i.e., when oxidative phosphorylation takes place. More than 30 years after the development of the method, here we aim to put the spotlight on it and to emphasize its versatile applicability in the most varied pathophysiological conditions, reviewing all the studies, in which we were able to observe what really happened in the cell thanks to the use of the "ATP detecting system" allowing the functional activity of the ANT-mediated ADP/ATP exchange to be measured.
    Keywords:  ATP detecting system; adenine nucleotide translocator; disease; mitochondria; physiological role; transport
  19. Circulation. 2021 Apr 28.
      Background: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially, but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional and functional adaption of human and murine mononuclear phagocytes (MNP). Methods: Using Seahorse technology, pulsed stable isotope-resolved metabolomics and enzyme activity assays we characterize the central carbon metabolism and mitochondrial function of human and murine MNP under HS in vitro. HS as well as pharmacologic uncoupling of the electron transport chain (ETC) under normal salt (NS) is used to analyze mitochondrial function on immune cell activation and function (as determined by E.coli killing and CD4+ T cell migration capacity). In two independent clinical studies we analyze the impact of a HS diet over two weeks (NCT02509962) and short-term salt challenge by a single meal (NCT04175249) on mitochondrial function of human monocytes in vivo. Results: Extracellular sodium was taken up into the intracellular compartment followed by the inhibition of mitochondrial respiration in murine and human macrophages (MΦ). Mechanistically, HS reduces mitochondrial membrane potential, ETC complex II activity, oxygen consumption, and ATP production independently of the polarization status of MΦ. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like MΦ and diminished CD4+ T cell migration in HS-treated M2-like MΦ. Pharmacologic uncoupling of the ETC under NS phenocopies HS-induced transcriptional changes and bactericidal function of human and murine MNP. Clinically, also in vivo rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both, a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of ̃x = 2mM and ̃x = 2.3mM, respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. Conclusions: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. While these functional changes might help to resolve bacterial infections, a shift towards pro-inflammation could accelerate inflammatory CVD.
    Keywords:  bacterial killing; complex II; mitochondrial respiration; salt
  20. Cancers (Basel). 2021 Apr 20. pii: 1980. [Epub ahead of print]13(8):
      Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis, high morbidity and mortality concerning with lack of effective diagnosis and high postoperative recurrence. Similar with other cancers, HCC cancer cells have to alter their metabolism to adapt to the changing requirements imposed by the environment of the growing tumor. In less vascularized regions of tumor, cancer cells experience hypoxia and nutrient starvation. Here, we show that HCC undergoes a global metabolic reprogramming during tumor growth. A combined proteomics and metabolomics analysis of paired peritumoral and tumor tissues from 200 HCC patients revealed liver-specific metabolic reprogramming and metabolic alterations with increasing tumor sizes. Several proteins and metabolites associated with glycolysis, the tricarboxylic acid cycle and pyrimidine synthesis were found to be differentially regulated in serum, tumor and peritumoral tissue with increased tumor sizes. Several prognostic metabolite biomarkers involved in HCC metabolic reprogramming were identified and integrated with clinical and pathological data. We built and validated this combined model to discriminate against patients with different recurrence risks. An integrated and comprehensive metabolomic analysis of HCC is provided by our present work. Metabolomic alterations associated with the advanced stage of the disease and poor clinical outcomes, were revealed. Targeting cancer metabolism may deliver effective therapies for HCC.
    Keywords:  NMR spectroscopy; hepatocellular carcinoma; metabolomics; predictive model; proteomics
  21. Cancers (Basel). 2021 Apr 01. pii: 1653. [Epub ahead of print]13(7):
      Head and neck squamous cell carcinoma (HNSCC) is characterized by high rates of mortality and treatment-related morbidity, underscoring the urgent need for innovative and safe treatment strategies and diagnosis practices. Mitochondrial dysfunction is a hallmark of cancer and can lead to the accumulation of tricarboxylic acid cycle intermediates, such as succinate, which function as oncometabolites. In addition to its role in cancer development through epigenetic events, succinate is an extracellular signal transducer that modulates immune response, angiogenesis and cell invasion by activating its cognate receptor SUCNR1. Here, we explored the potential value of the circulating succinate and related genes in HNSCC diagnosis and prognosis. We determined the succinate levels in the serum of 66 pathologically confirmed, untreated patients with HNSCC and 20 healthy controls. We also surveyed the expression of the genes related to succinate metabolism and signaling in tumoral and nontumoral adjacent tissue and in normal mucosa from 50 patients. Finally, we performed immunohistochemical analysis of SUCNR1 in mucosal samples. The results showed that the circulating levels of succinate were higher in patients with HNSCC than in the healthy controls. Additionally, the expression of SUCNR1, HIF-1α, succinate dehydrogenase (SDH) A, and SDHB was higher in the tumor tissue than in the matched normal mucosa. Consistent with this, immunohistochemical analysis revealed an increase in SUCNR1 protein expression in tumoral and nontumoral adjacent tissue. High SUCNR1 and SDHA expression levels were associated with poor locoregional control, and the locoregional recurrence-free survival rate was significantly lower in patients with high SUCNR1 and SDHA expression than in their peers with lower levels (77.1% [95% CI: 48.9-100.0] vs. 16.7% [95% CI: 0.0-44.4], p = 0.018). Thus, the circulating succinate levels are elevated in HNSCC and high SUCNR1/SDHA expression predicts poor locoregional disease-free survival, identifying this oncometabolite as a potentially valuable noninvasive biomarker for HNSCC diagnosis and prognosis.
    Keywords:  head and neck cancer; metabolism; oncometabolite; prognosis; succinate; succinate receptor; treatment
  22. Oncol Lett. 2021 Jun;21(6): 461
      Oral tongue squamous cell carcinoma (OTSCC) is a highly malignant type of tumor. The 5-year survival rate of patients with advanced tongue squamous cell carcinoma is only ~50%. Pyruvate kinase M2 (PKM2) is the key rate-limiting enzyme of glycolysis, maintaining the Warburg effect in tumor cells. The present study aimed to investigate the relationship between PKM2 expression and the poor prognosis of patients with OTSCC and to determine oral squamous carcinoma tumor cell proliferation and apoptosis. Reverse transcription-quantitative (RT-q) PCR, western blotting and immunohistochemistry were used to analyze the expression levels of PKM2 in OTSCC, and the clinicopathological characteristics and prognosis of patients with OTSCC were further analyzed by statistical analysis. The results from RT-qPCR and immunohistochemistry demonstrated that PKM2 was upregulated in OTSCC tissues and highly expressed in advanced stage OTSCC tissues compared with paired adjacent tissues and lower stage OTSCC tissues. Patients with OTSCC and high PKM2 expression had shorter overall survival (OS) compared with those with low PKM2 expression. Furthermore, high expression of PKM2 was significantly associated with Tumor-Node-Metastasis (TNM) stage. TNM stage and PKM2 expression were independent predictive factors for OS in patients with OTSCC. In addition, PKM2 knockdown inhibited the proliferation and increased the apoptosis of oral squamous carcinoma tumor cells. Furthermore, PKM2 knockdown could regulate the expression of cell cycle and apoptosis-related proteins by activating Hippo signaling pathway, as confirmed by the decreased expression of yes-associated protein 1 (YAP), Bcl-2 and Ki-67 and the increased expression of large tumor suppressor kinase 1, phosphorylated YAP and Bax. Taken together, the findings from this study demonstrated that PKM2 may be considered as a potential target for the diagnosis and treatment of OTSCC.
    Keywords:  Hippo pathway; apoptosis; oral tongue squamous cell carcinoma; proliferation; pyruvate kinase M2
  23. Life (Basel). 2021 Apr 08. pii: 325. [Epub ahead of print]11(4):
      Human diseases range from gene-associated to gene-non-associated disorders, including age-related diseases, neurodegenerative, neuromuscular, cardiovascular, diabetic diseases, neurocognitive disorders and cancer. Mitochondria participate to the cascades of pathogenic events leading to the onset and progression of these diseases independently of their association to mutations of genes encoding mitochondrial protein. Under physiological conditions, the mitochondrial ATP synthase provides the most energy of the cell via the oxidative phosphorylation. Alterations of oxidative phosphorylation mainly affect the tissues characterized by a high-energy metabolism, such as nervous, cardiac and skeletal muscle tissues. In this review, we focus on human diseases caused by altered expressions of ATP synthase genes of both mitochondrial and nuclear origin. Moreover, we describe the contribution of ATP synthase to the pathophysiological mechanisms of other human diseases such as cardiovascular, neurodegenerative diseases or neurocognitive disorders.
    Keywords:  ATP synthase; human disease; mitochondria
  24. Trends Cancer. 2021 Apr 22. pii: S2405-8033(21)00081-9. [Epub ahead of print]
      The mitochondrial electron transport chain (ETC) has been an attractive target for cancer therapy due to its essentiality for tumor growth. Krall et al. found that under ETC dysfunction, a decrease in asparagine limits cancer cell proliferation and activates the integrated stress response, creating a therapeutically exploitable metabolic vulnerability.
  25. Elife. 2021 04 27. pii: e59809. [Epub ahead of print]10
      Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.
    Keywords:  D. melanogaster; cancer biology; cell death; methionine; nutrition; oncogene; proliferation
  26. Elife. 2021 Apr 27. pii: e65108. [Epub ahead of print]10
      Adiponectin is essential for the regulation of tissue substrate utilization and systemic insulin sensitivity. Clinical studies have suggested a positive association of circulating adiponectin with healthspan and lifespan. However, the direct effects of adiponectin on promoting healthspan and lifespan remain unexplored. Here, we are using an adiponectin null mouse and a transgenic adiponectin overexpression model. We directly assessed the effects of circulating adiponectin on the aging process and found that adiponectin null mice display exacerbated age-related glucose and lipid metabolism disorders. Moreover, adiponectin null mice have a significantly shortened lifespan on both chow and high-fat diet (HFD). In contrast, a transgenic mouse model with elevated circulating adiponectin levels has a dramatically improved systemic insulin sensitivity, reduced age-related tissue inflammation and fibrosis, and a prolonged healthspan and median lifespan. These results support a role of adiponectin as an essential regulator for healthspan and lifespan.
    Keywords:  cell biology; mouse
  27. Antioxidants (Basel). 2021 Apr 08. pii: 570. [Epub ahead of print]10(4):
      Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with few therapeutic options. The identification of new promising targets is, therefore, an urgent need. Using available transcriptomic datasets, we first found that Peroxiredoxin-1 gene (PRDX1) expression was significantly increased in human pancreatic tumors, but not in the other gastrointestinal cancers; its high expression correlated with shortened patient survival. We confirmed by immunostaining on mouse pancreata the increased Peroxiredoxin-I protein (PRX-I) expression in pancreatic neoplastic lesions and PDAC. To question the role of PRX-I in pancreatic cancer, we genetically inactivated its expression in multiple human PDAC cell lines, using siRNA and CRISPR/Cas9. In both strategies, PRX-I ablation led to reduced survival of PDAC cells. This was mainly due to an increase in the production of reactive oxygen species (ROS), accumulation of oxidative DNA damage (i.e., 8-oxoguanine), and cell cycle blockade at G2/M. Finally, we found that PRX-I ablation disrupts the autophagic flux in PDAC cells, which is essential for their survival. This proof-of-concept study supports a pro-oncogenic role for PRX-I in PDAC.
    Keywords:  ROS; antioxidants; autophagy; cell cycle; pancreatic cancer
  28. Sensors (Basel). 2021 Apr 25. pii: 3017. [Epub ahead of print]21(9):
      Electric cell-substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 μM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 μM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.
    Keywords:  ECIS; capacitance; high frequency; micromotion; mitochondria; wound-healing migration
  29. Front Cell Dev Biol. 2021 ;9 653322
      The phenomenon of mitochondria donation is found in various tissues of humans and animals and is attracting increasing attention. To date, numerous studies have described the transfer of mitochondria from stem cells to injured cells, leading to increased ATP production, restoration of mitochondria function, and rescue of recipient cells from apoptosis. Mitochondria transplantation is considered as a novel therapeutic approach for the treatment of mitochondrial diseases and mitochondrial function deficiency. Mitochondrial dysfunction affects cells with high energy needs such as neural, skeletal muscle, heart, and liver cells and plays a crucial role in type 2 diabetes, as well as Parkinson's, Alzheimer's diseases, ischemia, stroke, cancer, and age-related disorders. In this review, we summarize recent findings in the field of mitochondria donation and mechanism of mitochondria transfer between cells. We review the existing clinical trials and discuss advantages and disadvantages of mitochondrial transplantation strategies based on the injection of stem cells, isolated functional mitochondria, or EVs containing mitochondria.
    Keywords:  cell fusion; extracellular vesicles; isolated mitochondria; mitochondria donation; mitochondria transplantation; tunneling nanotubes
  30. Int J Mol Sci. 2021 Apr 24. pii: 4461. [Epub ahead of print]22(9):
      Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.
    Keywords:  ANT1 mutations; mitochondrial diseases; yeast model
  31. Cancers (Basel). 2021 Apr 15. pii: 1914. [Epub ahead of print]13(8):
      Mitochondria are well known to participate in multiple aspects of tumor formation and progression. They indeed can alter the susceptibility of cells to engage regulated cell death, regulate pro-survival signal transduction pathways and confer metabolic plasticity that adapts to specific tumor cell demands. Interestingly, a relatively poorly explored aspect of mitochondria in neoplastic disease is their contribution to the characteristic genomic instability that underlies the evolution of the disease. In this review, we summarize the known mechanisms by which mitochondrial alterations in cancer tolerate and support the accumulation of DNA mutations which leads to genomic instability. We describe recent studies elucidating mitochondrial responses to DNA damage as well as the direct contribution of mitochondria to favor the accumulation of DNA alterations.
    Keywords:  ROS; apoptosis; calcium; genomic instability; mitochondria; mitophagy; p53; tumor progression
  32. Cell Prolif. 2021 May 01. e13034
      OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.MATERIALS AND METHODS: Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.
    RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N).
    CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.
    Keywords:  OPTN; PINK1; embryonic stem cells; mitochondria; mitophagy; reprogramming
  33. Antioxidants (Basel). 2021 Apr 20. pii: 627. [Epub ahead of print]10(4):
      Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.
    Keywords:  S-peroxiredoxinylation; cysteine sulfenic acid; hydrogen peroxide; peroxiredoxin; protein thiol oxidation; redox proteomics; redox relay; redox signaling; thiol disulfide exchange
  34. Cancers (Basel). 2021 Apr 13. pii: 1852. [Epub ahead of print]13(8):
      Oncogenic mutations in RAS family genes arise frequently in metastatic human cancers. Here we developed new mouse and cellular models of oncogenic HrasG12V-driven undifferentiated pleomorphic sarcoma metastasis and of KrasG12D-driven pancreatic ductal adenocarcinoma metastasis. Through analyses of these cells and of human oncogenic KRAS-, NRAS- and BRAF-driven cancer cell lines we identified that resistance to single MEK inhibitor and ERK inhibitor treatments arise rapidly but combination therapy completely blocks the emergence of resistance. The prior evolution of resistance to either single agent frequently leads to resistance to dual treatment. Dual MEK inhibitor plus ERK inhibitor therapy shows anti-tumor efficacy in an HrasG12V-driven autochthonous sarcoma model but features of drug resistance in vivo were also evident. Array-based kinome activity profiling revealed an absence of common patterns of signaling rewiring in single or double MEK and ERK inhibitor resistant cells, showing that the development of resistance to downstream signaling inhibition in oncogenic RAS-driven tumors represents a heterogeneous process. Nonetheless, in some single and double MEK and ERK inhibitor resistant cell lines we identified newly acquired drug sensitivities. These may represent additional therapeutic targets in oncogenic RAS-driven tumors and provide general proof-of-principle that therapeutic vulnerabilities of drug resistant cells can be identified.
    Keywords:  ERK inhibitor; MEK inhibitor; drug resistance; metastasis; mouse tumor model; oncogenic RAS; pancreatic ductal adenocarcinoma; undifferentiated pleomorphic sarcoma
  35. Cancer Metab. 2021 Apr 30. 9(1): 20
      Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate (OAA), serving to replenish the tricarboxylic acid (TCA) cycle. In nonmalignant tissue, PC plays an essential role in controlling whole-body energetics through regulation of gluconeogenesis in the liver, synthesis of fatty acids in adipocytes, and insulin secretion in pancreatic β cells. In breast cancer, PC activity is linked to pulmonary metastasis, potentially by providing the ability to utilize glucose, fatty acids, and glutamine metabolism as needed under varying conditions as cells metastasize. PC enzymatic activity appears to be of particular importance in cancer cells that are unable to utilize glutamine for anaplerosis. Moreover, PC activity also plays a role in lipid metabolism and protection from oxidative stress in cancer cells. Thus, PC activity may be essential to link energy substrate utilization with cancer progression and to enable the metabolic flexibility necessary for cell resilience to changing and adverse conditions during the metastatic process.
    Keywords:  Energy metabolism; Metastasis; Pyruvate carboxylase
  36. Mitochondrion. 2021 Apr 21. pii: S1567-7249(21)00057-X. [Epub ahead of print]
      Our laboratory has demonstrated that functional N-methyl-D-aspartate-like receptors are present on neuronal mitochondria (NMDAm). This novel site gates the influx of Ca2+ and causes a several-fold increase in ATP levels. Although elevations in ATP in other cell types have been linked to increases in mitochondrial Ca2+, it has not been established whether the same holds true for calcium uptake via NMDAm. In this study, we have investigated the effect of NMDAm activation on a variety of bioenergetic parameters. Our findings suggest that mitochondrial bioenergetics are not only modulated by NMDAm activation in a Ca2+-dependent but also in a Ca2+-independent manner.
    Keywords:  ATP; Bioenergetics; Calcium; Mitochondria; N-methyl-D-aspartate; Respiration
  37. Cancers (Basel). 2021 Apr 01. pii: 1662. [Epub ahead of print]13(7):
      Mitochondria are involved in the development and acquisition of a malignant phenotype in hematological cancers. Recently, their role in the pathogenesis of multiple myeloma (MM) has been suggested to be therapeutically explored. MYC is a master regulator of b-cell malignancies such as multiple myeloma, and its activation is known to deregulate mitochondrial function. We investigated the impact of mitochondrial activity on the distinct entities of the disease and tested the efficacy of the mitochondrial inhibitor, tigecycline, to overcome MM proliferation. COXII expression, COX activity, mitochondrial mass, and mitochondrial membrane potential demonstrated a progressive increase of mitochondrial features as the disease progresses. In vitro and in vivo therapeutic targeting using the mitochondrial inhibitor tigecycline showed promising efficacy and cytotoxicity in monotherapy and combination with the MM frontline treatment bortezomib. Overall, our findings demonstrate how mitochondrial activity emerges in MM transformation and disease progression and the efficacy of therapies targeting these novel vulnerabilities.
    Keywords:  MYC; mitochondria; multiple myeloma; tigecycline
  38. Oncogene. 2021 Apr 29.
      Mitochondrial oxidative phosphorylation (OXPHOS) is a vital regulator of tumor metastasis. However, the mechanisms governing OXPHOS to facilitate tumor metastasis remain unclear. In this study, we discovered that arginine 21(R21) and lysine 108 (K108) of mitochondrial ribosomal protein S23 (MRPS23) was methylated by the protein arginine methyltransferase 7 (PRMT7) and SET-domain-containing protein 6 (SETD6), respectively. R21 methylation accelerated the poly-ubiquitin-dependent degradation of MRPS23 to a low level. The MRPS23 degradation inhibited OXPHOS with elevated mtROS level, which consequently increased breast cancer cell invasion and metastasis. In contrast, K108 methylation increased MRPS23 stability, and K108 methylation coordinated with R21 methylation to maintain a low level of MRPS23, which was in favor of supporting breast cancer cell survival through regulating OXPHOS. Consistently, R21 and K108 methylation was correlated with malignant breast carcinoma. Significantly, our findings unveil a unique mechanism of controlling OXPHOS by arginine and lysine methylation and point to the impact of the PRMT7-SETD6-MRPS23 axis during breast cancer metastasis.
  39. Nature. 2021 Apr 26.
      The N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A writer METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but its true therapeutic importance is still unknown5-7. Here we present the identification and characterization of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) and its co-crystal structure bound to METTL3/METTL14. Treatment with STM2457 leads to reduced AML growth, and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various AML mouse models, specifically targeting key stem-cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA modifying enzymes represents a promising new avenue for anti-cancer therapy.
  40. BMC Cancer. 2021 Apr 30. 21(1): 481
      BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms.METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells.
    RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment.
    CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.
    Keywords:  Cancer cell lines; Cancer therapy; Dichloroacetate; Drug combination; HIF-1α inhibition; Metabolism; PX-478; Synergism