bims-mibica 2020-05-31 papers

bims-mibica

Biomed News

on Mitochondrial bioenergetics in cancer

Issue of 2020‒05‒31
thirty-five papers selected by
Kelsey Fisher-Wellman
East Carolina University

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.
Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines.
High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions.
An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.
Signal transducer and activator of transcription 5a (STAT5a) represses mitochondrial gene expression through direct binding to mitochondrial DNA.
Causative Role for Defective Expression of MIEAP in Accumulation of Mitochondria in Thyroid Oncocytic Cell Tumors.
TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels.
Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.
Functional changes induced by caloric restriction in cardiac and skeletal muscle mitochondria.
First-in-class candidate therapeutics that target mitochondria and effectively prevent cancer cell metastasis: mitoriboscins and TPP compounds.
MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.
Heterogeneity of Metabolic Vulnerability in Imatinib -Resistant Gastrointestinal Stromal Tumor.
Bcl2 inhibitor ABT737 reverses the Warburg effect via the Sirt3-HIF1α axis to promote oxidative stress-induced apoptosis in ovarian cancer cells.
Mitochondria participate in chemoresistance to cisplatin in human ovarian cancer cells.
Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.
SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2.
Systemic effects of mitochondrial stress.
Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids.
Mitochondrial Light Switches: Optogenetic Approaches to Control Metabolism.
Mitochondrial diseases in adults.
The mouse heart mitochondria N terminome provides insights into ClpXP-mediated proteolysis.
Premature synaptic mitochondrial dysfunction in the hippocampus during aging contributes to memory loss.
BH3 mimetics selectively eliminate chemotherapy-induced senescent cells and improve response in TP53 wild-type breast cancer.
TFAM depletion overcomes hepatocellular carcinoma resistance to doxorubicin and sorafenib through AMPK activation and mitochondrial dysfunction.
Nanoparticle delivery of a pH-sensitive prodrug of doxorubicin and a mitochondrial targeting VES-H8R8 synergistically kill multi-drug resistant breast cancer cells.
Molecular Choreography of Acute Exercise.
MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer.
MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men.
Selective Induction of Cellular Toxicity and Anti-tumor Efficacy by N-Methylpiperazinyl Diarylidenylpiperidone and its Pro-nitroxide Conjugate through ROS-mediated Mitochondrial Dysfunction and G2/M Cell-cycle Arrest in Human Pancreatic Cancer.
Nitrate attenuates HFD-induced glucose intolerance in association with reduced epididymal adipose tissue inflammation and mitochondrial ROS emission.
MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis.
Acetylation promotes BCAT2 degradation to suppress BCAA catabolism and pancreatic cancer growth.
Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.
UCP2 silencing in glioblastoma reduces cell proliferation and invasiveness by inhibiting p38 MAPK pathway.
Interplay between MTOR and GPX4 signaling modulates autophagy-dependent ferroptotic cancer cell death.

Nat Commun. 2020 May 29. 11(1): 2682

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Sonia Alcalá, Patricia Sancho, Paola Martinelli, Diego Navarro, Coral Pedrero, Laura Martín-Hijano, Sandra Valle, Julie Earl, Macarena Rodríguez-Serrano, Laura Ruiz-Cañas, Katerin Rojas, Alfredo Carrato, Laura García-Bermejo, Miguel Ángel Fernández-Moreno, Patrick C Hermann, Bruno Sainz.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

DOI: https://doi.org/10.1038/s41467-020-16395-2
Int J Mol Sci. 2020 May 25. pii: E3731. [Epub ahead of print]21(10):

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines.

Consuelo Ripoll, Mar Roldan, Rafael Contreras-Montoya, Juan J Diaz-Mochon, Miguel Martin, Maria J Ruedas-Rama, Angel Orte.

  The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes.

Keywords:  FLIM microscopy; cancer metabolism; intracellular sensors; nanosensing; tumoral metabolism

DOI:  https://doi.org/10.3390/ijms21103731
Commun Biol. 2020 May 29. 3(1): 271

High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions.

Andrew Charles Little, Ilya Kovalenko, Laura Elaine Goo, Hanna Sungok Hong, Samuel Andrew Kerk, Joel Anthony Yates, Vinee Purohit, David Benner Lombard, Sofia Diana Merajver, Costas Andreas Lyssiotis.

Metabolic flux technology with the Seahorse bioanalyzer has emerged as a standard technique in cellular metabolism studies, allowing for simultaneous kinetic measurements of respiration and glycolysis. Methods to extend the utility and versatility of the metabolic flux assay would undoubtedly have immediate and wide-reaching impacts. Herein, we describe a platform that couples the metabolic flux assay with high-content fluorescence imaging to simultaneously provide means for normalization of respiration data with cell number; analyze cell cycle distribution; and quantify mitochondrial content, fragmentation state, membrane potential, and mitochondrial reactive oxygen species. Integration of fluorescent dyes directly into the metabolic flux assay generates a more complete data set of mitochondrial features in a single assay. Moreover, application of this integrated strategy revealed insights into mitochondrial function following PGC1a and PRC1 inhibition in pancreatic cancer and demonstrated how the Rho-GTPases impact mitochondrial dynamics in breast cancer.

DOI: https://doi.org/10.1038/s42003-020-0988-z
Bioorg Chem. 2020 May 18. pii: S0045-2068(20)30548-4. [Epub ahead of print]100 103935

An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.

Viviana Donoso-Bustamante, Edgar A Borrego, Yareli Schiaffino-Bustamante, Denisse A Gutiérrez, Juan Pablo Millas-Vargas, Sebastián Fuentes-Retamal, Pablo Correa, Ileana Carrillo, Renato J Aguilera, Dante Miranda, Ignacio Chávez-Báez, Rodrigo Pulgar, Félix A Urra, Armando Varela-Ramírez, Ramiro Araya-Maturana.

  Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial β-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.

Keywords:  Cancer cells; Fatty acids; Hydroquinone; Leukemia; Metabolic adaptation; Mitochondria; β-oxidation

DOI:  https://doi.org/10.1016/j.bioorg.2020.103935
Biochem Biophys Res Commun. 2020 May 20. pii: S0006-291X(20)30893-7. [Epub ahead of print]

Signal transducer and activator of transcription 5a (STAT5a) represses mitochondrial gene expression through direct binding to mitochondrial DNA.

Fu-Yu Chueh, Ying-Ling Chang, Shin-Yu Wu, Chao-Lan Yu.

  Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors essential for cytokine signaling. Our previous study showed that interleukin-3 (IL-3) induced STAT5 translocation to mitochondria and binding to mitochondrial DNA (mtDNA) in vitro. In this report, we further demonstrated in vivo binding of endogenous STAT5a to mtDNA transcriptional control region and reduced gene expression from all three mtDNA promoters after IL-3 stimulation. To specifically define the function of mitochondrial STAT5a, we generated mitochondrial-targeting wild-type and mutant STAT5a proteins. Compared with non-targeting STAT5a, mitochondrial-targeting wild-type STAT5a significantly reduced mitochondrial gene expression in transfected HEK293 cells. The level of attenuation was amplified in cells expressing constitutively active STAT5a, but abrogated in cells expressing DNA-binding-defective STAT5a. STAT5a-mediated repression of mtDNA expression also positively correlated with STAT5a binding to the E2 subunit of pyruvate dehydrogenase complex (PDC-E2), both a gate-keeping metabolic enzyme and a component of mtDNA nucleoid in mitochondrial matrix. Metabolic shift away from mitochondrial respiration is known in many cytokine-stimulated cells and cancer cells. STAT5a-mediated repression of mitochondrial gene expression and its interaction with PDC-E2 may provide important insights into its underlying mechanisms.

Keywords:  Electron transport chain; Interleukin-3; Metabolism; Mitochondria; STAT5

DOI:  https://doi.org/10.1016/j.bbrc.2020.04.152
Cancer Sci. 2020 May 27.

Causative Role for Defective Expression of MIEAP in Accumulation of Mitochondria in Thyroid Oncocytic Cell Tumors.

Zhanna Mussazhanova, Mika Shimamura, Tomomi Kurashige, Masahiro Ito, Masahiro Nakashima, Yuji Nagayama.

  Oncocytic cell tumor of the thyroid is composed of large polygonal cells with eosinophilic cytoplasm that is rich in mitochondria. These tumors frequently have the mutations in mitochondrial DNA encoding the mitochondrial electron transport system complex I. However, the mechanism for accumulation of abnormal mitochondria is unknown. A non-canonical mitophagy system has recently been identified, and mitochondria-eating protein (MIEAP) plays a key role in this system. We therefore hypothesized that accumulation of abnormal mitochondria could be attributed to defective MIEAP expression in these tumors. We first show that MIEAP was expressed in all the conventional thyroid follicular adenomas (FAs)/adenomatous goiters (AGs) but not in oncocytic FAs/AGs, while its expression was defective not only oncocytic thyroid cancers but also in the majority of conventional thyroid cancers. MIEAP expression was not correlated with methylation status of 5'-untranslated region of the gene. Our functional analysis demonstrated that exogenously induced MIEAP but not PARK2 reduced the amounts of abnormal mitochondria, as demonstrated by decreased reactive oxygen species levels, mitochondrial DNA/nuclear DNA ratios and cytoplasmic acidification. Therefore, together with previous studies showing that impaired mitochondrial function triggers compensatory mitochondrial biogenesis that causes an increase in the amounts of mitochondria, we would like to conclude that, in oncocytic cell tumors of the thyroid, increased abnormal mitochondria cannot be efficiently eliminated because of a loss of MIEAP expression, i.e., impaired MIEAP-mediated non-canonical mitophagy.

Keywords:  ; mitochondria; mitochondria-eating protein; mitophagy; oncocytic cell tumor; thyroid

DOI:  https://doi.org/10.1111/cas.14501
Cancers (Basel). 2020 May 22. pii: E1329. [Epub ahead of print]12(5):

TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels.

Wookyeom Yang, Ha-Yeon Shin, Hanbyoul Cho, Joon-Yong Chung, Eun-Ju Lee, Jae-Hoon Kim, Eun-Suk Kang.

  TOM40 is a channel-forming subunit of translocase, which is essential for the movement of proteins into the mitochondria. We found that TOM40 was highly expressed in epithelial ovarian cancer (EOC) cells at both the transcriptional and translational levels; its expression increased significantly during the transformation from normal ovarian epithelial cells to EOC (p < 0.001), and TOM40 expression negatively correlated with disease-free survival (Hazard ratio = 1.79, 95% Confidence inerval 1.16-2.78, p = 0.009). TOM40 knockdown decreased proliferation in several EOC cell lines and reduced tumor burden in an in vivo xenograft mouse model. TOM40 expression positively correlated with intracellular adenosine triphosphate (ATP) levels. The low ATP and high reactive oxygen species (ROS) levels increased the activity of AMP-activated protein kinase (AMPK) in TOM40 knockdown EOC cells. However, AMPK activity did not correlate with declined cell growth in TOM40 knockdown EOC cells. We found that metformin, first-line therapy for type 2 diabetes, effectively inhibited the growth of EOC cell lines in an AMPK-independent manner by inhibiting mitochondria complex I. In conclusion, TOM40 positively correlated with mitochondrial activities, and its association enhances the proliferation of ovarian cancer. Also, metformin is an effective therapeutic option in TOM40 overexpressed ovarian cancer than normal ovarian epithelium.

Keywords:  TOM40; epithelial ovarian cancer; metformin; mitochondria

DOI:  https://doi.org/10.3390/cancers12051329
Elife. 2020 May 28. pii: e49178. [Epub ahead of print]9

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.

Eran Mick, Denis V Titov, Owen S Skinner, Rohit Sharma, Alexis A Jourdain, Vamsi K Mootha.

  Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.

Keywords:  ATF4; GCN2; genetics; genomics; human; human biology; integrated stress response; medicine; metabolism; mitochondria; mouse; p53

DOI:  https://doi.org/10.7554/eLife.49178
J Bioenerg Biomembr. 2020 May 27.

Functional changes induced by caloric restriction in cardiac and skeletal muscle mitochondria.

Julian David C Serna, Camille C Caldeira da Silva, Alicia J Kowaltowski.

  Caloric restriction (CR) is widely known to increase life span and resistance to different types of injuries in several organisms. We have previously shown that mitochondria from livers or brains of CR animals exhibit higher calcium uptake rates and lower sensitivity to calcium-induced mitochondrial permeability transition (mPT), an event related to the resilient phenotype exhibited by these organs. Given the importance of calcium in metabolic control and cell homeostasis, we aimed here to uncover possible changes in mitochondrial calcium handling, redox balance and bioenergetics in cardiac and skeletal muscle mitochondria in response to six months of CR. Unexpectedly, we found that CR does not alter the susceptibility to mPT in muscle (cardiac or skeletal), nor calcium uptake rates. Despite the lack in changes in calcium transport properties, CR consistently decreased respiration in the presence of ATP synthesis in heart and soleus muscle. In heart, such changes were accompanied by a decrease in respiration in the absence of ATP synthesis, lower maximal respiratory rates and a reduced rate of hydrogen peroxide release. Hydrogen peroxide release was unaltered by CR in skeletal muscle. No changes were observed in inner membrane potentials and respiratory control ratios. Together, these results highlight the tissue-specific bioenergetic and ion transport effects induced by CR, demonstrating that resilience against calcium-induced mPT is not present in all tissues.

Keywords:  Bioenergetics; Caloric Restriction; Heart; Mitochondrial Permeability Transition; Reactive Oxygen Species; Soleus Muscle

DOI:  https://doi.org/10.1007/s10863-020-09838-4
Aging (Albany NY). 2020 May 24. 12

First-in-class candidate therapeutics that target mitochondria and effectively prevent cancer cell metastasis: mitoriboscins and TPP compounds.

Béla Ózsvári, Federica Sotgia, Michael P Lisanti.

  Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence, distant metastasis and drug-resistance, in the vast majority of cancer patients. Therefore, there is an urgent need to identify new drugs that can target and eradicate CSCs. To identify new molecular targets that are unique to CSCs, we previously compared MCF7 2D-monolayers with 3D-mammospheres, which are enriched in CSCs. We observed that 25 mitochondrial-related proteins were >100-fold over-expressed in 3D-mammospheres. Here, we used these 25 proteins to derive short gene signatures to predict distant metastasis (in N=1,395 patients) and tumor recurrence (in N=3,082 patients), by employing a large collection of transcriptional profiling data from ER(+) breast cancer patients. This analysis resulted in a 4-gene signature for predicting distant metastasis, with a hazard ratio of 1.91-fold (P=2.2e-08). This provides clinical evidence to support a role for CSC mitochondria in metastatic dissemination. Next, we employed a panel of mitochondrial inhibitors, previously shown to target mitochondria and selectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells. Remarkably, these five mitochondrial inhibitors had only minor effects or no effect on MDA-MB-231 tumor formation, but preferentially and selectively inhibited tumor cell metastasis, without causing significant toxicity. Mechanistically, all five mitochondrial inhibitors have been previously shown to induce ATP-depletion in cancer cells. Since 3 of these 5 inhibitors were designed to target the large mitochondrial ribosome, we next interrogated whether genes encoding the large mitochondrial ribosomal proteins (MRPL) also show prognostic value in the prediction of distant metastasis in both ER(+) and ER(-) breast cancer patients. Interestingly, gene signatures composed of 6 to 9 MRPL mRNA-transcripts were indeed sufficient to predict distant metastasis, tumor recurrence and Tamoxifen resistance. These gene signatures could be useful as companion diagnostics to assess which patients may benefit most from anti-mito-ribosome therapy. Overall, our studies provide the necessary proof-of-concept, and in vivo functional evidence, that mitochondrial inhibitors can successfully and selectively target the biological process of cancer cell metastasis. Ultimately, we envision that mitochondrial inhibitors could be employed to develop new treatment protocols, for clinically providing metastasis prophylaxis, to help prevent poor clinical outcomes in cancer patients.

Keywords:  breast cancer; cancer stem-like cells (CSCs); metastasis prophylaxis; mitochondrial inhibitors; treatment failure

DOI:  https://doi.org/10.18632/aging.103336
J Cell Physiol. 2020 May 26.

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.

Alexandre Houzelle, Dennis Dahlmans, Emmani B M Nascimento, Gert Schaart, Johanna A Jörgensen, Esther Moonen-Kornips, Sander Kersten, Xu Wang, Joris Hoeks.

  Using an unbiased high-throughput microRNA (miRNA)-silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA-204-5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA-204-5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA-204-5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC-1α (3.71-fold; p < .01), predicted as an miR-204-5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA-204-5p silencing in C2C12 myotubes. Silencing of miRNA-204-5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA-204-5p silencing. Finally, miRNA-204-5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA-204-5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA-204-5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA-204-5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

Keywords:  C2C12; microRNA; mitochondria; mitophagy; skeletal muscle

DOI:  https://doi.org/10.1002/jcp.29797
Cells. 2020 May 26. pii: E1333. [Epub ahead of print]9(6):

Heterogeneity of Metabolic Vulnerability in Imatinib -Resistant Gastrointestinal Stromal Tumor.

Wen-Kuan Huang, Jiwei Gao, Ziqing Chen, Hao Shi, Juan Yuan, Huanhuan L Cui, Chun-Nan Yeh, Robert Bränström, Catharina Larsson, Shuijie Li, Weng-Onn Lui.

  Metabolic reprogramming is a hallmark of cancer cells in response to targeted therapy. Decreased glycolytic activity with enhanced mitochondrial respiration secondary to imatinib has been shown in imatinib-sensitive gastrointestional stromal tumors (GIST). However, the role of energy metabolism in imatinib-resistant GIST remains poorly characterized. Here, we investigated the effect of imatinib treatment on glycolysis and oxidative phosphorylation (OXPHOS), as well as the effect of inhibition of these energy metabolisms on cell viability in imatinib-resistant and -sensitive GIST cell lines. We observed that imatinib treatment increased OXPHOS in imatinib-sensitive, but not imatinib-resistant, GIST cells. Imatinib also reduced the expression of mitochondrial biogenesis activators (peroxisome proliferator-activated receptor coactivator-1 alpha (PGC1α), nuclear respiratory factor 2 (NRF2), and mitochondrial transcription factor A (TFAM)) and mitochondrial mass in imatinib-sensitive GIST cells. Lower TFAM levels were also observed in imatinib-sensitive GISTs than in tumors from untreated patients. Using the Seahorse system, we observed bioenergetics diversity among the GIST cell lines. One of the acquired resistant cell lines (GIST 882R) displayed a highly metabolically active phenotype with higher glycolysis and OXPHOS levels compared with the parental GIST 882, while the other resistant cell line (GIST T1R) had a similar basal glycolytic activity but lower mitochondrial respiration than the parental GIST T1. Further functional assays demonstrated that GIST 882R was more vulnerable to glycolysis inhibition than GIST 882, while GIST T1R was more resistant to OXPHOS inhibition than GIST T1. These findings highlight the diverse energy metabolic adaptations in GIST cells that allow them to survive upon imatinib treatment and reveal the potential of targeting the metabolism for GIST therapy.

Keywords:  energy metabolism; gastrointestinal stromal tumor; imatinib resistance; mitochondrial biogenesis

DOI:  https://doi.org/10.3390/cells9061333
Life Sci. 2020 May 26. pii: S0024-3205(20)30596-8. [Epub ahead of print] 117846

Bcl2 inhibitor ABT737 reverses the Warburg effect via the Sirt3-HIF1α axis to promote oxidative stress-induced apoptosis in ovarian cancer cells.

Delu Dong, Yuan Dong, Jiaying Fu, Shengyao Lu, Chunli Yuan, Meihui Xia, Liankun Sun.

  AIMS: Compared to normal cells, tumor cells maintain higher concentrations of reactive oxygen species (ROS) to support proliferation, invasion, and metastasis. Chemotherapeutic drugs often induce tumor cell apoptosis by increasing intracellular ROS concentrations to highly toxic levels. ABT737, which inhibits the apoptosis regulator B cell lymphoma 2 (Bcl2), increases the sensitivity of ovarian cancer cells to chemotherapeutic drugs by regulating the glucose metabolism, but the underlying mechanisms remain unclear. Therefore, we aimed to determine whether ABT737 promoted H2O2-induced tumor cell apoptosis by reversing glycolysis in ovarian cancer cells.MAIN METHODS: SKOV3 ovarian cancer cells were treated with H2O2, ABT737, or both. Cell viability was compared using methyl thiazolyl tetrazolium (MTT), and flow cytometry was used to detect differences in apoptosis, ROS, and mitochondrial membrane potential. The relative expression levels of proteins associated with apoptosis and the glucose metabolism were measured using immunoblotting. Finally, glucose uptake and lactate secretion were measured using kits and compared.
KEY FINDINGS: ABT737 downregulated proteins associated with glucose uptake (GLUT1) and glycolysis (LHDA, PKM2 and HK2) via the Sirt3-HIF1α axis, reducing glucose uptake and lactate secretion in SKOV3 cells. This reversed glycolysis in the tumor cells, and promoted H2O2-induced apoptosis.
SIGNIFICANCE: The Bcl2 inhibitor ABT737 enhanced the anti-tumor effect of oxidative stress by reversing the Warburg effect in ovarian cancer cells, providing powerful theoretical support for further clinical applications of Bcl2 inhibitors.

Keywords:  ABT737; Apoptosis; B-cell lymphoma 2; Glycolysis; Oxidative stress; Warburg effect

DOI:  https://doi.org/10.1016/j.lfs.2020.117846
Mol Cancer Res. 2020 May 29. pii: molcanres.1145.2019. [Epub ahead of print]

Mitochondria participate in chemoresistance to cisplatin in human ovarian cancer cells.

Luca X Zampieri, Debora Grasso, Caroline Bouzin, Davide Brusa, Rodrigue Rossignol, Pierre Sonveaux.

Ovarian cancer is an aggressive disease that affects about 300,000 patients worldwide, with a yearly death count of about 185,000. Following surgery, treatment involves adjuvant or neoadjuvant administration of taxane with platinum compounds cisplatin or carboplatin, which alkylate DNA through the same chemical intermediates. However, although platinum-based therapy can cure patients in a number of cases, a majority of them discontinues treatment owing to side effects and to the emergence of resistance. In this study, we focused on resistance to cisplatin and investigated whether metabolic changes could be involved. As models, we used matched pairs of cisplatin-sensitive (SKOV-3 and COV-362) and cisplatin-resistant (SKOV-3-R and COV-362-R) human ovarian carcinoma cells that were selected in vitro following exposure to increasing doses of the chemotherapy. Metabolic comparison revealed that resistant cells undergo a shift towards a more oxidative metabolism. The shift goes along with a reorganization of the mitochondrial network, with a generally increased mitochondrial compartment. More functional mitochondria in cisplatin-resistant compared to cisplatin-sensitive cells were associated to enzymatic changes affecting either the electron transport chain (SKOV-3/SKOV-3-R model) or mitochondrial coupling (COV-362/COV-362-R model). Our findings further indicate that the preservation of functional mitochondria in these cells could be due to an increased mitochondrial turnover rate, suggesting mitophagy inhibition as a potential strategy to tackle cisplatin-resistant human ovarian cancer progression. Implications: Besides classical mechanisms related to drug efflux and target modification, we report that preserving functional mitochondria is a strategy used by human ovarian cancer cells to resist to cisplatin chemotherapy.

DOI: https://doi.org/10.1158/1541-7786.MCR-19-1145
FASEB J. 2020 May 28.

Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.

Geeta Rao, Brennah Murphy, Anindya Dey, Shailendra Kumar Dhar Dwivedi, Yushan Zhang, Ram Vinod Roy, Prabir Chakraborty, Resham Bhattacharya, Priyabrata Mukherjee.

  Mutations in the human cystathionine beta synthase (CBS) gene are known to cause endothelial dysfunction responsible for cardiovascular and neurovascular diseases. CBS is the predominant hydrogen sulfide (H2 S)-producing enzyme in endothelial cells (ECs). Recently, H2 S was shown to attenuate ROS and improve mitochondrial function. Mitochondria are metabolic organelles that actively transform their ultrastructure to mediate their function. Therefore, we questioned whether perturbation of CBS/H2 S activity could drive mitochondrial dysfunction via mitochondrial dynamics in ECs. Here we demonstrate that silencing CBS induces mitochondria fragmentation, attenuates efficient oxidative phosphorylation, and decreases EC function. Mechanistically, CBS silencing significantly elevates ROS production, thereby leading to reduced mitofusin 2 (MFN2) expression, decouple endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death. These defects were significantly rescued by the treatment of H2 S donors. Taken together our data highlights a novel signaling axis that mechanistically links CBS with mitochondrial function and ER-mitochondrial tethering and could be considered as a new therapeutic approach for the intervention of EC dysfunction-related pathologies.

Keywords:  endothelial cells; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; mitophagy

DOI:  https://doi.org/10.1096/fj.202000173R
Sci Rep. 2020 May 26. 10(1): 8677

SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2.

Katarína Smolková, Jitka Špačková, Klára Gotvaldová, Aleš Dvořák, Alena Křenková, Martin Hubálek, Blanka Holendová, Libor Vítek, Petr Ježek.

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

DOI: https://doi.org/10.1038/s41598-020-65351-z
EMBO Rep. 2020 May 24. e50094

Systemic effects of mitochondrial stress.

Raz Bar-Ziv, Theodore Bolas, Andrew Dillin.

  Multicellular organisms are complex biological systems, composed of specialized tissues that require coordination of the metabolic and fitness state of each component. In the cells composing the tissues, one central organelle is the mitochondrion, a compartment essential for many energetic and fundamental biological processes. Beyond serving these functions, mitochondria have emerged as signaling hubs in biological systems, capable of inducing changes to the cell they are in, to cells in distal tissues through secreted factors, and to overall animal physiology. Here, we describe our current understanding of these communication mechanisms in the context of mitochondrial stress. We focus on cellular mechanisms that deal with perturbations to the mitochondrial proteome and outline recent advances in understanding how local perturbations can affect distal tissues and animal physiology in model organisms. Finally, we discuss recent findings of these responses associated with metabolic and age-associated diseases in mammalian systems, and how they may be employed as diagnostic and therapeutic tools.

Keywords:  aging; mitochondria; stress

DOI:  https://doi.org/10.15252/embr.202050094
Biochem Biophys Res Commun. 2020 Jun 18. pii: S0006-291X(20)30833-0. [Epub ahead of print]527(1): 162-166

Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids.

Sivakama S Bharathi, Yuxun Zhang, Zhenwei Gong, Radhika Muzumdar, Eric S Goetzman.

  Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway.

Keywords:  Acyl-CoA dehydrogenase; Acyl-CoA oxidase; Dicarboxylic fatty acids; Fatty acid oxidation; Mitochondria; Peroxisomes

DOI:  https://doi.org/10.1016/j.bbrc.2020.04.105
FEBS J. 2020 May 27.

Mitochondrial Light Switches: Optogenetic Approaches to Control Metabolism.

Brandon J Berry, Andrew P Wojtovich.

  Developing new technologies to study metabolism is increasingly important as metabolic disease prevalence increases. Mitochondria control cellular metabolism and dynamic changes in mitochondrial function are associated with metabolic abnormalities in cardiovascular disease, cancer, and obesity. However, a lack of precise and reversible methods to control mitochondrial function has prevented moving from association to causation. Recent advances in optogenetics have addressed this challenge, and mitochondrial function can now be precisely controlled in vivo using light. A class of genetically-encoded, light-activated membrane channels and pumps has addressed mechanistic questions that promise to provide new insights into how cellular metabolism downstream of mitochondrial function contributes to disease. Here, we highlight emerging reagents - mitochondria-targeted light-activated cation channels or proton pumps - to decrease or increase mitochondrial activity upon light exposure, a technique we refer to as mitochondrial light switches, or mtSWITCH . The mtSWITCH technique is broadly applicable, as energy availability and metabolic signaling are conserved aspects of cellular function and health. Here, we outline the use of these tools in diverse cellular models of disease. We review the molecular details of each optogenetic tool, summarize the results obtained with each, and outline best practices for using optogenetic approaches to control mitochondrial function and downstream metabolism.

Keywords:  AMPK; Parkinson’s; apoptosis; bioenergetics; calcium signaling; diabetes; hypoxia; mitophagy

DOI:  https://doi.org/10.1111/febs.15424
J Intern Med. 2020 Jun;287(6): 592-608

Mitochondrial diseases in adults.

C La Morgia, A Maresca, L Caporali, M L Valentino, V Carelli.

  Mitochondrial medicine is a field that expanded exponentially in the last 30 years. Individually rare, mitochondrial diseases as a whole are probably the most frequent genetic disorder in adults. The complexity of their genotype-phenotype correlation, in terms of penetrance and clinical expressivity, natural history and diagnostic algorithm derives from the dual genetic determination. In fact, in addition to the about 1.500 genes encoding mitochondrial proteins that reside in the nuclear genome (nDNA), we have the 13 proteins encoded by the mitochondrial genome (mtDNA), for which 22 specific tRNAs and 2 rRNAs are also needed. Thus, besides Mendelian genetics, we need to consider all peculiarities of how mtDNA is inherited, maintained and expressed to fully understand the pathogenic mechanisms of these disorders. Yet, from the initial restriction to the narrow field of oxidative phosphorylation dysfunction, the landscape of mitochondrial functions impinging on cellular homeostasis, driving life and death, is impressively enlarged. Finally, from the clinical standpoint, starting from the neuromuscular field, where brain and skeletal muscle were the primary targets of mitochondrial dysfunction as energy-dependent tissues, after three decades virtually any subspecialty of medicine is now involved. We will summarize the key clinical pictures and pathogenic mechanisms of mitochondrial diseases in adults.

Keywords:  mitochondria; mitochondrial diseases; mtDNA; neurology; neuromuscular disorders

DOI:  https://doi.org/10.1111/joim.13064
Mol Cell Proteomics. 2020 May 28. pii: mcp.RA120.002082. [Epub ahead of print]

The mouse heart mitochondria N terminome provides insights into ClpXP-mediated proteolysis.

Eduard Hofsetz, Fatih Demir, Karolina Szczepanowska, Alexandra Kukat, Jayachandran Kizhakkedathu, Aleksandra Trifunovic, Pitter F Huesgen.

  The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp-/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondrial N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Keywords:  Affinity proteomics; Degradomics*; Mitochondria function or biology; Proteolysis*; Substrate identification

DOI:  https://doi.org/10.1074/mcp.RA120.002082
Redox Biol. 2020 May 05. pii: S2213-2317(20)30412-2. [Epub ahead of print]34 101558

Premature synaptic mitochondrial dysfunction in the hippocampus during aging contributes to memory loss.

Margrethe A Olesen, Angie K Torres, Claudia Jara, Michael P Murphy, Cheril Tapia-Rojas.

  Aging is a process characterized by cognitive impairment and mitochondrial dysfunction. In neurons, these organelles are classified as synaptic and non-synaptic mitochondria depending on their localization. Interestingly, synaptic mitochondria from the cerebral cortex accumulate more damage and are more sensitive to swelling than non-synaptic mitochondria. The hippocampus is fundamental for learning and memory, synaptic processes with high energy demand. However, it is unknown if functional differences are found in synaptic and non-synaptic hippocampal mitochondria; and whether this could contribute to memory loss during aging. In this study, we used 3, 6, 12 and 18 month-old (mo) mice to evaluate hippocampal memory and the function of both synaptic and non-synaptic mitochondria. Our results indicate that recognition memory is impaired from 12mo, whereas spatial memory is impaired at 18mo. This was accompanied by a differential function of synaptic and non-synaptic mitochondria. Interestingly, we observed premature dysfunction of synaptic mitochondria at 12mo, indicated by increased ROS generation, reduced ATP production and higher sensitivity to calcium overload, an effect that is not observed in non-synaptic mitochondria. In addition, at 18mo both mitochondrial populations showed bioenergetic defects, but synaptic mitochondria were prone to swelling than non-synaptic mitochondria. Finally, we treated 2, 11, and 17mo mice with MitoQ or Curcumin (Cc) for 5 weeks, to determine if the prevention of synaptic mitochondrial dysfunction could attenuate memory loss. Our results indicate that reducing synaptic mitochondrial dysfunction is sufficient to decrease age-associated cognitive impairment. In conclusion, our results indicate that age-related alterations in ATP produced by synaptic mitochondria are correlated with decreases in spatial and object recognition memory and propose that the maintenance of functional synaptic mitochondria is critical to prevent memory loss during aging.

Keywords:  Aging; Hippocampus; Memory; Mitochondria; Non-synaptic; Synaptic

DOI:  https://doi.org/10.1016/j.redox.2020.101558
Cell Death Differ. 2020 May 26.

BH3 mimetics selectively eliminate chemotherapy-induced senescent cells and improve response in TP53 wild-type breast cancer.

Ashkan Shahbandi, Sonia G Rao, Ashlyn Y Anderson, Wesley D Frey, Joy O Olayiwola, Nathan A Ungerleider, James G Jackson.

TP53 wild-type breast tumors rarely undergo a complete pathological response after chemotherapy treatment. These patients have an extremely poor survival rate and studies show these tumors preferentially undergo senescence instead of apoptosis. These senescent cells persist after chemotherapy and secrete cytokines and chemokines comprising the senescence associated secretory phenotype, which promotes survival, proliferation, and metastasis. We hypothesized that eliminating senescent tumor cells would improve chemotherapy response and extend survival. Previous studies have shown "senolytic" agents selectively kill senescent normal cells, but their efficacy in killing chemotherapy-induced senescent cancer cells is unknown. We show that ABT-263, a BH3 mimetic that targets antiapoptotic proteins BCL2/BCL-XL/BCL-W, had no effect on proliferating cells, but rapidly and selectively induced apoptosis in a subset of chemotherapy-treated cancer cells, though sensitivity required days to develop. Low NOXA expression conferred resistance to ABT-263 in some cells, necessitating additional MCL1 inhibition. Gene editing confirmed breast cancer cells relied on BCL-XL or BCL-XL/MCL1 for survival in senescence. In a mouse model of breast cancer, ABT-263 treatment following chemotherapy led to apoptosis, greater tumor regression, and longer survival. Our results reveal cancer cells that have survived chemotherapy by entering senescence can be eliminated using BH3 mimetic drugs that target BCL-XL or BCL-XL/MCL1. These drugs could help minimize residual disease and extend survival in breast cancer patients that otherwise have a poor prognosis and are most in need of improved therapies.

DOI: https://doi.org/10.1038/s41418-020-0564-6
Gene. 2020 May 24. pii: S0378-1119(20)30476-5. [Epub ahead of print] 144807

TFAM depletion overcomes hepatocellular carcinoma resistance to doxorubicin and sorafenib through AMPK activation and mitochondrial dysfunction.

Ying Zhu, Jianguo Xu, Wei Hu, Fang Wang, Yan Zhou, Wen Xu, Wei Gong, Lichun Shao.

  Mitochondrial transcription factor A (TFAM), which is required for mitochondrial DNA (mtDNA) transcription, has been linked to metabolic changes that contribute to tumorigenesis and chemoresistance. In this work, we investigated the expression pattern and role of TFAM in hepatocellular carcinoma (HCC). TFAM expression level is similar in 18 out of 20 paired normal liver and HCC tissues with only 2 HCC tissues showing 1.8-fold increase in TFAM. Similar phenomenon was observed in HCC cell lines compared to normal liver lines. Interestingly, TFAM expression is upregulated in resistant HCC cells regardless of the differential TFAM expression level in their parental lines and mechanism of resistance. TFAM depletion led to inhibition of growth and survival but not migration, and sensitization to doxorubicin and sorafenib treatment, through AMPK activation, reduction of nucleoside triphosphates and mitochondrial respiration in HCC cells. In addition, we demonstrated that resistant HCC cell lines were more sensitive to TFAM inhibition than parental lines, and this might be due to the increased mitochondrial biogenesis in resistant HCC cell lines. Our work reveals the preferential role of TFAM in HCC cell response to standard of care drugs, which suggests a potential sensitizing therapeutic target for HCC treatment.

Keywords:  Chemoresistance; Hepatocellular carcinoma; Mitochondrial biogenesis; TFAM

DOI:  https://doi.org/10.1016/j.gene.2020.144807
Sci Rep. 2020 May 26. 10(1): 8726

Nanoparticle delivery of a pH-sensitive prodrug of doxorubicin and a mitochondrial targeting VES-H8R8 synergistically kill multi-drug resistant breast cancer cells.

Petro Czupiel, Vianney Delplace, Molly Shoichet.

Multi-drug resistance (MDR) remains a major obstacle in cancer treatment while being heavily dependent on mitochondrial activity and drug efflux. We previously demonstrated that cationic lipids, such as the vitamin E succinate modified octahistidine-octaarginine (VES-H8R8) conjugate, target mitochondria, resulting in depolarized mitochondria and inhibited drug efflux in MDR breast cancer cells. We hypothesized that the effective cell uptake, efflux inhibition, and mitochondrial depolarization properties of VES-H8R8 would synergistically enhance the toxicity of a pH-sensitive prodrug of doxorubicin (pDox) when co-encapsulated in nanoparticles (NPs). pDox was successfully synthesized and validated for pH-sensitive release from NPs under lysosome-mimicking, acidic conditions. The synergistic effect of VES-H8R8 and pDox was confirmed against MDR breast cancer cells in vitro. Importantly, synergism was only observed when VES-H8R8 and pDox were co-encapsulated in a single nanoparticulate system. The synergistic mechanism was investigated, confirming superior pDox uptake and retention, Pgp efflux inhibition, mitochondrial depolarization, and enhanced induction of ROS, and apoptosis. This work demonstrates the translational potential of doubly-loaded NPs co-encapsulating pDox with VES-H8R8 to synergistically kill MDR breast cancer cells.

DOI: https://doi.org/10.1038/s41598-020-65450-x
Cell. 2020 May 28. pii: S0092-8674(20)30508-0. [Epub ahead of print]181(5): 1112-1130.e16

Molecular Choreography of Acute Exercise.

Kévin Contrepois, Si Wu, Kegan J Moneghetti, Daniel Hornburg, Sara Ahadi, Ming-Shian Tsai, Ahmed A Metwally, Eric Wei, Brittany Lee-McMullen, Jeniffer V Quijada, Songjie Chen, Jeffrey W Christle, Mathew Ellenberger, Brunilda Balliu, Shalina Taylor, Matthew G Durrant, David A Knowles, Hani Choudhry, Melanie Ashland, Amir Bahmani, Brooke Enslen, Myriam Amsallem, Yukari Kobayashi, Monika Avina, Dalia Perelman, Sophia Miryam Schüssler-Fiorenza Rose, Wenyu Zhou, Euan A Ashley, Stephen B Montgomery, Hassan Chaib, Francois Haddad, Michael P Snyder.

  Acute physical activity leads to several changes in metabolic, cardiovascular, and immune pathways. Although studies have examined selected changes in these pathways, the system-wide molecular response to an acute bout of exercise has not been fully characterized. We performed longitudinal multi-omic profiling of plasma and peripheral blood mononuclear cells including metabolome, lipidome, immunome, proteome, and transcriptome from 36 well-characterized volunteers, before and after a controlled bout of symptom-limited exercise. Time-series analysis revealed thousands of molecular changes and an orchestrated choreography of biological processes involving energy metabolism, oxidative stress, inflammation, tissue repair, and growth factor response, as well as regulatory pathways. Most of these processes were dampened and some were reversed in insulin-resistant participants. Finally, we discovered biological pathways involved in cardiopulmonary exercise response and developed prediction models revealing potential resting blood-based biomarkers of peak oxygen consumption.

Keywords:  cardiopulmonary exercise testing; fitness; insulin resistance; multi-omics; outlier analysis; peak VO(2); physical activity; predictive analytics; systems biology; time-series analysis

DOI:  https://doi.org/10.1016/j.cell.2020.04.043
Cancer Cell. 2020 May 13. pii: S1535-6108(20)30215-4. [Epub ahead of print]

MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer.

Arnaud Augert, Haritha Mathsyaraja, Ali H Ibrahim, Brian Freie, Michael J Geuenich, Pei-Feng Cheng, Sydney P Alibeckoff, Nan Wu, Joseph B Hiatt, Ryan Basom, Adi Gazdar, Lucas B Sullivan, Robert N Eisenman, David MacPherson.

  Small cell lung cancer (SCLC) is a highly aggressive and lethal neoplasm. To identify candidate tumor suppressors we applied CRISPR/Cas9 gene inactivation screens to a cellular model of early-stage SCLC. Among the top hits was MAX, the obligate heterodimerization partner for MYC family proteins that is mutated in human SCLC. Max deletion increases growth and transformation in cells and dramatically accelerates SCLC progression in an Rb1/Trp53-deleted mouse model. In contrast, deletion of Max abrogates tumorigenesis in MYCL-overexpressing SCLC. Max deletion in SCLC resulted in derepression of metabolic genes involved in serine and one-carbon metabolism. By increasing serine biosynthesis, Max-deleted cells exhibit resistance to serine depletion. Thus, Max loss results in metabolic rewiring and context-specific tumor suppression.

Keywords:  CRISPR-Cas9 genetic screens; MAX; MYC; SCLC; Transcriptional regulation; cancer; mouse model; serine and one-carbon metabolism; small cell lung cancer; tumor suppressor genes

DOI:  https://doi.org/10.1016/j.ccell.2020.04.016
Eur J Appl Physiol. 2020 May 26.

MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men.

Toan Pham, Caitlin L MacRae, Sophie C Broome, Randall F D'souza, Ravi Narang, Hsiang W Wang, Trevor A Mori, Anthony J R Hickey, Cameron J Mitchell, Troy L Merry.

  PURPOSE: Excess production of reactive oxygen species (ROS) from the mitochondria can promote mitochondrial dysfunction and has been implicated in the development of a range of chronic diseases. As such there is interest in whether mitochondrial-targeted antioxidant supplementation can attenuate mitochondrial-associated oxidative stress. We investigated the effect of MitoQ and CoQ10 supplementation on oxidative stress and skeletal muscle mitochondrial ROS levels and function in healthy middle-aged men.METHODS: Skeletal muscle and blood samples were collected from twenty men (50 ± 1 y) before and following six weeks of daily supplementation with MitoQ (20 mg) or CoQ10 (200 mg). High-resolution respirometry was used to determine mitochondrial respiration and H2O2 levels, markers of mitochondrial mass and antioxidant defences were measured in muscle samples and oxidative stress markers in urine and blood samples.
RESULTS: Both MitoQ and CoQ10 supplementation suppressed mitochondrial net H2O2 levels during leak respiration, while MitoQ also elevated muscle catalase expression. However, neither supplement altered urine F2-isoprostanes nor plasma TBARS levels. Neither MitoQ nor CoQ10 supplementation had a significant impact on mitochondrial respiration or mitochondrial density markers (citrate synthase, mtDNA/nDNA, PPARGC1A, OXPHOS expression).
CONCLUSION: Our results suggest that neither MitoQ and CoQ10 supplements impact mitochondrial function, but both can mildly suppress mitochondrial ROS levels in healthy middle-aged men, with some indication that MitoQ may be more effective than CoQ10.

Keywords:  Antioxidant; Mitochondria; Muscle; Oxidative stress; ROS

DOI:  https://doi.org/10.1007/s00421-020-04396-4
Cell Biochem Biophys. 2020 May 24.

Selective Induction of Cellular Toxicity and Anti-tumor Efficacy by N-Methylpiperazinyl Diarylidenylpiperidone and its Pro-nitroxide Conjugate through ROS-mediated Mitochondrial Dysfunction and G2/M Cell-cycle Arrest in Human Pancreatic Cancer.

Jesse M Mast, John W Hinds, Dan Tse, Katherine Axelrod, M Lakshmi Kuppusamy, Maciej M Kmiec, Balázs Bognár, Tamás Kálai, Periannan Kuppusamy.

  Pancreatic adenocarcinoma is an aggressive cancer with poor clinical prognosis and limited therapeutic options. There is a significant lack of effective, safe, and targeted therapies for successful treatment of pancreatic cancer. In this report, we describe the anticancer efficacy of two novel compounds, N-methylpiperazinyl diarylidenylpiperidone (L-2663) and its pro-nitroxide conjugate (HO-4589) evaluated on human pancreatic adenocarcinoma (AsPC-1) cell line and xenograft tumor in mice. Using flow cytometry, we determined the effect of the L-2663 and HO-4589 drugs in inducing mitochondrial toxicity, triggering cell-cycle arrest, and apoptosis. EPR spectroscopy was used to quantify cellular uptake, metabolic conversion and stability of HO-4589 in cells and in vivo monitoring of tumor oxygenation as a function of growth. The results established different antiproliferative efficacy of the L-2663 and HO-4589 compounds, with a targeted action on cancer cells while being less toxic to noncancerous cells. The study may have important implications in the future designs of safe and effective chemotherapeutic agents for the treatment of pancreatic cancer.

Keywords:  Diarylidenylpiperidone; Nitroxide; Pancreatic cancer; ROS; Tumor oxygen; Xenograft tumor

DOI:  https://doi.org/10.1007/s12013-020-00919-0
J Physiol. 2020 May 25.

Nitrate attenuates HFD-induced glucose intolerance in association with reduced epididymal adipose tissue inflammation and mitochondrial ROS emission.

Henver S Brunetta, Valerie Politis-Barber, Heather L Petrick, Kaitlyn M J H Dennis, Aleah J Kirsh, Pierre-Andre Barbeau, Everson A Nunes, Graham P Holloway.

  KEY POINTS: Dietary nitrate is a prominent therapeutic strategy to mitigate some metabolic deleterious effects related to obesity. Mitochondrial dysfunction is causally linked to adipose tissue inflammation and insulin resistance. Whole-body glucose tolerance is prevented by nitrate independent of body weight and energy expenditure. Dietary nitrate reduces epididymal adipose tissue inflammation and mitochondrial reactive oxygen species emission while preserving insulin signalling. Metabolic beneficial effects of nitrate consumption are associated with improvements in mitochondrial redox balance in hypertrophic adipose tissue.ABSTRACT: Evidence has accumulated to indicate that dietary nitrate alters energy expenditure and the metabolic derangements associated with a high-fat diet, however, the mechanism(s) of action remain incompletely elucidated. Therefore, we aimed to determine if dietary nitrate (4 mm sodium nitrate via drinking water) could prevent high-fat diet (HFD) mediated glucose intolerance in association with improved mitochondrial bioenergetics within both white (WAT) and brown (BAT) adipose tissue. HFD-feeding caused glucose intolerance (P < 0.05) and increased body weight. As a result of higher body weight, energy expenditure increased proportionally. HFD-fed mice displayed greater mitochondrial uncoupling and a 2-fold increase in UCP-1 content within BAT. Within epididymal adipose tissue (eWAT), HFD increased cell size (i.e. hypertrophy), mitochondrial H2 O2 emission, oxidative stress, JNK phosphorylation, leucocyte infiltration, and induced insulin resistance. Remarkably, dietary nitrate consumption attenuated and/or mitigated all these responses, including rendering mitochondria more coupled within BAT, and normalizing mitochondrial H2 O2 emission and insulin-mediated Akt-Thr308 phosphorylation within eWAT. Intriguingly, the positive effects of dietary nitrate appear to be independent of eWAT mitochondrial respiratory capacity and content. Altogether, these data suggest that dietary nitrate attenuates the development of HFD-induced insulin resistance in association with attenuating WAT inflammation and redox balance, independent of changes within either WAT or BAT mitochondrial respiratory capacity/content. This article is protected by copyright. All rights reserved.

Keywords:  insulin resistance; mitochondrial function; nitrate; nutrition; obesity

DOI:  https://doi.org/10.1113/JP279455
Nat Metab. 2020 Mar;2(3): 270-277

MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis.

Chien-Kuang Cornelia Ding, Joshua Rose, Tianai Sun, Jianli Wu, Po-Han Chen, Chao-Chieh Lin, Wen-Hsuan Yang, Kai-Yuan Chen, Hana Lee, Emily Xu, Sarah Tian, Jadesola Akinwuntan, Jinshi Zhao, Ziqiang Guan, Pei Zhou, Jen-Tsan Chi.

Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosis-inducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

DOI: https://doi.org/10.1038/s42255-020-0181-1
Signal Transduct Target Ther. 2020 May 29. 5(1): 70

Acetylation promotes BCAT2 degradation to suppress BCAA catabolism and pancreatic cancer growth.

Ming-Zhu Lei, Xu-Xu Li, Ye Zhang, Jin-Tao Li, Fan Zhang, Yi-Ping Wang, Miao Yin, Jia Qu, Qun-Ying Lei.

Pancreatic ductal adenocarcinoma (PDAC) is well-known for inefficient early diagnosis, with most patients diagnosed at advanced stages. Increasing evidence indicates that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with an increased risk of pancreatic cancer. Branched-chain amino acid transaminase 2 (BCAT2) is an important enzyme in BCAA catabolism that reversibly catalyzes the initial step of BCAA degradation to branched-chain acyl-CoA. Here, we show that BCAT2 is acetylated at lysine 44 (K44), an evolutionarily conserved residue. BCAT2 acetylation leads to its degradation through the ubiquitin-proteasome pathway and is stimulated in response to BCAA deprivation. cAMP-responsive element-binding (CREB)-binding protein (CBP) and SIRT4 are the acetyltransferase and deacetylase for BCAT2, respectively. CBP and SIRT4 bind to BCAT2 and control the K44 acetylation level in response to BCAA availability. More importantly, the K44R mutant promotes BCAA catabolism, cell proliferation, and pancreatic tumor growth. Collectively, the data from our study reveal a previously unknown regulatory mechanism of BCAT2 in PDAC and provide a potential therapeutic target for PDAC treatment.

DOI: https://doi.org/10.1038/s41392-020-0168-0
Blood. 2020 May 26. pii: blood.2019001808. [Epub ahead of print]

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Rushdia Yusuf, Borja Saez, Azeem Sharda, Nick van Gastel, Vionnie Yu, Ninib Baryawno, Elizabeth W Scadden, Sanket S Acharya, Shrikanta Chattophadhyay, Cherrie Huang, Vasanthi Viswanathan, Dana S'aulis, Julien Cobert, David Brian Sykes, Mark A Keibler, Sudeshna Das, John N Hutchinson, Michael Churchill, Siddharta Mukherjee, Dongjun Lee, Francois Emile Mercier, John Doench, Lars Bullinger, David J Logan, Stuart Schreiber, Gregory Stephanopoulos, William B Rizzo, David T Scadden.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and generated from lipid peroxides underlying the non-caspase dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition, a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

DOI: https://doi.org/10.1182/blood.2019001808
Exp Cell Res. 2020 May 26. pii: S0014-4827(20)30356-6. [Epub ahead of print] 112110

UCP2 silencing in glioblastoma reduces cell proliferation and invasiveness by inhibiting p38 MAPK pathway.

Shuai Wu, Chen Luo, N U Farrukh Hameed, Ye Wang, Dongxiao Zhuang.

  Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane anion carrier and is emerging as a negative regulator of ROS production. Overexpression of UCP2 has been detected in various tumors, but its role in glioblastoma remains unclear. Using tissue microarrays and interrogations of public databases, we explored that the expression of UCP2 is upregulated in glioma, especially in GBM, and overexpression of UCP2 correlates with poor prognosis in glioma patients. To further reveal the role of UCP2 in glioma, UCP2-slienced cell lines (U251, U87MG and A172) by lentivirus were constructed to study how silenced UCP2 expression affects cellular functions in vitro, and tumorigenicity in vivo. RNA-Seq based genome and pathway analysis were performed to elucidate the underlying mechanisms of action of UCP2. Our results revealed that UCP2 silenced glioma cells show inhibited migration, invasiveness, clonogenicity, proliferation, promoted cell apoptosis in vitro, and weaker tumorigenicity in nude mice. Transcriptome analysis suggested a UCP2-dependent regulation of p38 MAPK (Mitogen-activated protein kinase) signaling networks, which was further validated by qRT-PCR and Western blot. Our research provides a new insight into the biological significance of UCP2 in glioma and its potential application in treatment and diagnosis.

Keywords:  Apoptosis; GBM; Invasiveness; Proliferation; UCP2; p38

DOI:  https://doi.org/10.1016/j.yexcr.2020.112110
Cancer Gene Ther. 2020 May 27.

Interplay between MTOR and GPX4 signaling modulates autophagy-dependent ferroptotic cancer cell death.

Yang Liu, Yuan Wang, Jiao Liu, Rui Kang, Daolin Tang.

Ferroptosis has become a topic of rapidly growing interest in recent years, and has possible therapy implications in cancer therapy. Although excessive autophagy may contribute to ferroptosis, its underlying molecular mechanism remains largely unknown. Here, we provide novel evidence that the interplay between the signals of mechanistic target of rapamycin kinase (MTOR) and glutathione peroxidase 4 (GPX4) modulates autophagy-dependent ferroptosis in human pancreatic cancer cells. Both the classical autophagy inducer rapamycin and the classical ferroptosis activator RSL3 can block MTOR activation and cause GPX4 protein degradation in human pancreatic cancer cells. Moreover, GPX4 plays an essential role in the inhibition of autophagy-dependent ferroptosis induced by rapamycin and RSL3. Consequently, GPX4 depletion by RNAi enhances the anticancer activity of rapamycin and RSL3 in vitro or in vivo. These findings not only increase our understanding of stress responses in cell death, but may also raise the possibility of developing new antitumor therapy targeting autophagy-dependent cell death.

DOI: https://doi.org/10.1038/s41417-020-0182-y