bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒07‒10
forty-two papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Oncogene. 2022 Jul 02.
      We previously found that lactic acidosis in the tumor environment was permissive to cancer cell surviving under glucose deprivation and demonstrated that neutralizing lactic acidosis restored cancer cell susceptibility to glucose deprivation. We then reported that alternate infusion of bicarbonate and anticancer agent into tumors via tumor feeding artery markedly enhanced the efficacy of transarterial chemoembolization (TACE) in the local control of hepatocellular carcinoma (HCC). Here we sought to further investigate the mechanism by which bicarbonate enhances the anticancer activity of TACE. We propose that interfering cellular pH by bicarbonate could induce a cascade of molecular events leading to cancer cell death. Alkalizing cellular pH by bicarbonate decreased pH gradient (ΔpH), membrane potential (ΔΨm), and proton motive force (Δp) across the inner membrane of mitochondria; disruption of oxidative phosphorylation (OXPHOS) due to collapsed Δp led to a significant increase in adenosine monophosphate (AMP), which activated the classical AMPK-mediated autophagy. Meanwhile, the autophagic flux was ultimately blocked by increased cellular pH, reduced OXPHOS, and inhibition of lysosomal proton pump in alkalized lysosome. Bicarbonate also induced persistent mitochondrial permeability (MPT) and damaged mitochondria. Collectively, this study reveals that interfering cellular pH may provide a valuable approach to treat cancer.
  2. Cancer Metab. 2022 Jul 04. 10(1): 10
      BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently.METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites.
    RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions.
    CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.
    Keywords:  Adaptation; Colorectal cancer; Glucose; Irinotecan; Metabolism; Warburg effect; p53
  3. Biochim Biophys Acta Mol Basis Dis. 2022 Jul 02. pii: S0925-4439(22)00152-1. [Epub ahead of print] 166481
      Mitochondrial-derived reactive oxygen species are important as antimicrobial agents and redox signals in pro-inflammatory macrophages. Macrophages produce superoxide in response to the TLR4 ligand LPS. However, the mechanism of LPS-induced superoxide generation is not fully understood. Superoxide is produced at complex I and complex III of the electron transport chain. Production of superoxide at either of these sites is highly dependent on the metabolic state of the cell which is dramatically altered by TLR4-induced metabolic reprogramming. This review will outline how metabolism impacts superoxide production in LPS-activated macrophages downstream of TLR4 signalling and address outstanding questions in this field.
    Keywords:  Complex I; Macrophages; Metabolism; Mitochondria; Reverse electron transport; Superoxide
  4. Leukemia. 2022 Jul 08.
      Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1b deletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1b deletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1b deletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9 cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.
  5. Pflugers Arch. 2022 Jul 02.
      Mitochondrial temperature is produced by various metabolic processes inside the mitochondria, particularly oxidative phosphorylation. It was recently reported that mitochondria could normally operate at high temperatures that can reach 50℃. The aim of this review is to identify mitochondrial temperature differences between normal cells and cancer cells. Herein, we discussed the different types of mitochondrial thermosensors and their advantages and disadvantages. We reviewed the studies assessing the mitochondrial temperature in cancer cells and normal cells. We shed the light on the factors involved in maintaining the mitochondrial temperature of normal cells compared to cancer cells.
    Keywords:  Heat shock proteins; Mitochondria; Mitochondrial temperature; Oxidative phosphorylation; Uncoupling proteins
  6. Carcinogenesis. 2022 Jul 04. pii: bgac060. [Epub ahead of print]
      Sphingolipid metabolism plays an important role in the formation of cellular membranes and is associated with malignant potential and chemosensitivity of cancer cells. Sphingolipid degradation depends on multiple lysosomal glucosidases. We focused on acid β-glucosidase (GBA), a lysosomal enzyme the deficiency of which is related to mitochondrial dysfunction. We analyzed the function of GBA in pancreatic ductal adenocarcinoma (PDAC). Human PDAC cell lines (PANC-1, BxPC-3, and AsPC-1) were examined under conditions of GBA knockdown via the short interfering RNA (siRNA) method. We assessed the morphological changes, GBA enzyme activity, GBA protein expression, cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), and mitophagy flux of PDAC cells. The GBA protein level and enzyme activity differed among cell lines. GBA knockdown suppressed cell proliferation and induced apoptosis, especially in PANC-1 and BxPC-3 cells, with low GBA enzyme activity. GBA knockdown also decreased the MMP and impaired mitochondrial clearance. This impaired mitochondrial clearance further induced dysfunctional mitochondria accumulation and ROS generation in PDAC cells, inducing apoptosis. The antiproliferative effects of the combination of GBA suppression and gemcitabine were higher than those of gemcitabine alone. These results showed that GBA suppression exerts a significant antitumor effect and may have therapeutic potential in the clinical treatment of PDAC.
  7. Nat Rev Mol Cell Biol. 2022 Jul 08.
      Mitochondrial energetic adaptations encompass a plethora of conserved processes that maintain cell and organismal fitness and survival in the changing environment by adjusting the respiratory capacity of mitochondria. These mitochondrial responses are governed by general principles of regulatory biology exemplified by changes in gene expression, protein translation, protein complex formation, transmembrane transport, enzymatic activities and metabolite levels. These changes can promote mitochondrial biogenesis and membrane dynamics that in turn support mitochondrial respiration. The main regulatory components of mitochondrial energetic adaptation include: the transcription coactivator peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC1α) and associated transcription factors; mTOR and endoplasmic reticulum stress signalling; TOM70-dependent mitochondrial protein import; the cristae remodelling factors, including mitochondrial contact site and cristae organizing system (MICOS) and OPA1; lipid remodelling; and the assembly and metabolite-dependent regulation of respiratory complexes. These adaptive molecular and structural mechanisms increase respiration to maintain basic processes specific to cell types and tissues. Failure to execute these regulatory responses causes cell damage and inflammation or senescence, compromising cell survival and the ability to adapt to energetically demanding conditions. Thus, mitochondrial adaptive cellular processes are important for physiological responses, including to nutrient availability, temperature and physical activity, and their failure leads to diseases associated with mitochondrial dysfunction such as metabolic and age-associated diseases and cancer.
  8. Cell Stem Cell. 2022 Jul 07. pii: S1934-5909(22)00253-3. [Epub ahead of print]29(7): 1119-1134.e7
      Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.
    Keywords:  GCN2; amino acid; hematopoietic stem cells; metabolism; nicotinamide riboside; oxidative phosphorylation; protein translation; proteostasis
  9. Cancers (Basel). 2022 Jun 27. pii: 3129. [Epub ahead of print]14(13):
      Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.
    Keywords:  cannabidiol; cannabinoids; electron transport chain complex proteins; glioblastoma cells; mitochondria; oxygen consumption rate; Δ9-tetrahydrocannabinol
  10. Autophagy. 2022 Jul 04.
      Cancer-associated fibroblasts (CAFs) are considered one of the most critical stromal cells that interact with pancreatic ductal adenocarcinoma (PDAC) and promote tumor growth, metastasis, and treatment resistance. Previous studies illustrated macroautophagy/autophagy contributes to CAF activation during tumor progression. Here in our study, we found that autophagy deficiency in CAFs impedes CAF activation by inhibiting proline biosynthesis and collagen production. Furthermore, we uncovered that autophagy promotes proline biosynthesis through mitophagy-mediated regulation of NADK2 (NAD kinase 2, mitochondrial), an enzyme responsible for production of mitochondrial NADP(H). Using an orthotopic mouse model of PDAC, we found that inhibiting mitophagy by targeting PRKN (parkin RBR E3 ubiquitin protein ligase) in the stroma reduced tumor weight. Thus, inhibition of CAFs mitophagy might be an attractive strategy for stroma-focused anti-cancer intervention.
    Keywords:  NADK2; autophagy; cancer-associated fibroblasts (CAFs); mitophagy; proline biosynthesis
  11. Int J Mol Sci. 2022 Jul 04. pii: 7419. [Epub ahead of print]23(13):
      Cancer cells switch their metabolism toward glucose metabolism to sustain their uncontrolled proliferation. Consequently, glycolytic intermediates are diverted into the pentose phosphate pathway (PPP) to produce macromolecules necessary for cell growth. The transcription regulator RIP140 controls glucose metabolism in tumor cells, but its role in cancer-associated reprogramming of cell metabolism remains poorly understood. Here, we show that, in human breast cancer cells and mouse embryonic fibroblasts, RIP140 inhibits the expression of the gene-encoding G6PD, the first enzyme of the PPP. RIP140 deficiency increases G6PD activity as well as the level of NADPH, a reducing cofactor essential for macromolecule synthesis. Moreover, G6PD knock-down inhibits the gain of proliferation observed when RIP140 expression is reduced. Importantly, RIP140-deficient cells are more sensitive to G6PD inhibition in cell proliferation assays and tumor growth experiments. Altogether, this study describes a novel role for RIP140 in regulating G6PD levels, which links its effect on breast cancer cell proliferation to metabolic rewiring.
    Keywords:  G6PD; RIP140; breast cancer; pentose phosphate pathway; transcription
  12. Comput Struct Biotechnol J. 2022 ;20 3059-3067
      Extrachromosomal circular DNA (eccDNA) of chromosomal origin is common in eukaryotic cells. Amplification of oncogenes on large eccDNA (ecDNA) can drive biological processes such as tumorigenesis, and identification of eccDNA by sequencing after removal of chromosomal DNA is therefore important for understanding their impact on the expressed phenotype. However, the circular mitochondrial DNA (mtDNA) might challenge the detection of eccDNA because the average somatic cell has hundreds of copies of mtDNA. Here we show that 61.2-99.5% of reads from eccDNA-enriched samples correspond to mtDNA in mouse tissues. We have developed a method to selectively remove mtDNA from total circular DNA by CRISPR/Cas9 guided cleavage of mtDNA with one single-guide RNA (sgRNA) or two sgRNAs followed by exonuclease degradation of the linearized mtDNA. Sequencing revealed that mtDNA reads were 85.9% ± 12.6% removed from eccDNA of 9 investigated mouse tissues. CRISPR/Cas9 cleavage also efficiently removed mtDNA from a human HeLa cell line and colorectal cancer samples. We identified up to 14 times more, and also larger eccDNA in CRISPR/Cas9 treated colorectal cancer samples than in untreated samples. We foresee that the method can be applied to effectively remove mtDNA from any eukaryotic species to obtain higher eccDNA yields.
    Keywords:  CRISPR/Cas9; ecDNA; eccDNA; mtDNA removal
  13. Nat Metab. 2022 Jul 04.
      Anti-programmed death-1 (PD-1) immunotherapy that aims to restore T cell activity in cancer patients frequently leads to immune-related adverse events such as colitis. However, the underlying mechanism is still elusive. Here, we find that Pdcd1-deficient mice exhibit disrupted gut microbiota and aggravated dextran sulfate sodium (DSS)-induced colitis. In addition to T cells, PD-1 is also substantially expressed in colonic lymphoid tissue inducer (LTi) cells. During DSS-induced colitis, LTi cell activation is accompanied by increased PD-1 expression, whereas PD-1 deficiency results in reduced interleukin-22 (IL-22) production by LTi cells and exacerbated inflammation. Mechanistically, activated LTi cells reprogram their metabolism toward carbohydrate metabolism and fatty acid synthesis, while fatty acid oxidation (FAO) is unchanged. However, PD-1 deficiency leads to significantly elevated FAO in LTi cells, which in turn attenuates their activation and IL-22 production. Consistently, FAO suppression efficiently restores IL-22 production in Pdcd1-/- LTi cells. Thus, our study provides unforeseen mechanistic insight into colitis occurrence during anti-PD-1 immunotherapy through LTi cell metabolic reconfiguration.
  14. J Biol Chem. 2022 Jun 29. pii: S0021-9258(22)00656-1. [Epub ahead of print] 102214
      Mitochondrial translation is a highly regulated process, and newly synthesized mitochondrial products must first associate with several nuclear-encoded auxiliary factors to form oxidative phosphorylation complexes. The output of mitochondrial products should therefore be in stoichiometric equilibrium with the nuclear-encoded products to prevent unnecessary energy expense or the accumulation of pro-oxidant assembly modules. In the mtDNA of Saccharomyces cerevisiae, COX1 encodes subunit 1 of the cytochrome c oxidase, and COB the central core of the cytochrome bc1 electron transfer complex; however, factors regulating the expression of these mitochondrial products are not well described. In this study, we identified Mrx9p as a new factor that controls COX1 and COB expression. We isolated MRX9 in a screen for mitochondrial factors that cause poor accumulation of newly synthesized Cox1p and compromised transition to the respiratory metabolism. Northern analyses indicated lower levels of COX1 and COB mature mRNAs accompanied by an accumulation of unprocessed transcripts in the presence of excess Mrx9p. Furthermore, in a strain devoid of mitochondrial introns, MRX9 overexpression did not affect COX1 and COB translation or respiratory adaptation, implying Mrx9p regulates processing of COX1 and COB RNAs. In addition, we found Mrx9p was localized in the mitochondrial inner membrane, facing the matrix, as a portion of it co-sedimented with mitoribosome subunits and its removal or overexpression altered Mss51p sedimentation. Finally, we showed accumulation of newly synthesized Cox1p in the absence of Mrx9p was diminished in cox14 null mutants. Taken together, these data indicate a regulatory role of Mrx9p in COX1 RNA processing.
    Keywords:  Saccharomyces cerevisiae; intron processing; mitochondrial translation
  15. Front Oncol. 2022 ;12 829007
      Objectives: Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with widely variable prognosis. For this reason, a more tailored-stratified approach for prognosis is urgently needed to improve the treatment success rates of AML patients.Methods: In the investigation of metabolic pattern in AML patients, we developed a metabolism-related prognostic model, which was consisted of metabolism-related gene pairs (MRGPs) identified by pairwise comparison. Furthermore, we analyzed the predictive ability and clinical significance of the prognostic model.
    Results: Given the significant differences in metabolic pathways between AML patients and healthy donors, we proposed a metabolism-related prognostic signature index (MRPSI) consisting of three MRGPs, which were remarkedly related with the overall survival of AML patients in the training set. The association of MRPSI with prognosis was also validated in two other independent cohorts, suggesting that high MRPSI score can identify patients with poor prognosis. The MRPSI and age were confirmed to be independent prognostic factors via multivariate Cox regression analysis. Furthermore, we combined MRPSI with age and constructed a composite metabolism-clinical prognostic model index (MCPMI), which demonstrated better prognostic accuracy in all cohorts. Stratification analysis and multivariate Cox regression analysis revealed that the MCPMI was an independent prognostic factor. By estimating the sensitivity of anti-cancer drugs in different AML patients, we selected five drugs that were more sensitive to patients in MCPMI-high group than those in MCPMI-low group.
    Conclusion: Our study provided an individualized metabolism-related prognostic model that identified high-risk patients and revealed new potential therapeutic drugs for AML patients with poor prognosis.
    Keywords:  MCPMI; MRPSI; acute myeloid leukemia; drug response; prognosis
  16. Cell Death Discov. 2022 Jul 05. 8(1): 307
      Metabolic remodeling is the fundamental molecular feature of malignant tumors. Cancer cells require sufficient energy supplies supporting their high proliferative rate. MTHFD2, a mitochondrial one-carbon metabolic enzyme, is dysregulated in several malignancies and may serve as a promising therapeutic candidate in cancer treatment. Here, our data confirmed that MTHFD2 gene and protein was upregulated in the cancerous tissues of LUAD patients and was correlated with a poor survival in LUAD. MTHFD2 was involved in lung cancer cell proliferation, migration, and apoptosis by mediating its downstream molecules, such as DNA helicases (MCM4 and MCM7), as well as ZEB1, Vimentin and SNAI1, which contributed to tumor cell growth and epithelial-to-mesenchymal transition (EMT) process. Moreover, we identified that miRNA-99a-3p appeared to be an upstream mediator directly regulating MTHFD2 and MCM4 expression. Moreover, specific inhibition of MTHFD2 functions by siRNA or a chemical compound, improved anti-tumor sensitivities induced by pemetrexed in LUAD. Taken together, our study revealed the underlying molecular mechanisms of MTHFD2 in regulating cell proliferation and identified a novel therapeutic strategy improving the treatment efficacies in LUAD.
  17. Nat Commun. 2022 Jul 08. 13(1): 3947
      Succinate dehydrogenase, which is known as mitochondrial complex II, has proven to be a fascinating machinery, attracting renewed and increased interest in its involvement in human diseases. Herein, we find that succinate dehydrogenase assembly factor 4 (SDHAF4) is downregulated in cardiac muscle in response to pathological stresses and in diseased hearts from human patients. Cardiac loss of Sdhaf4 suppresses complex II assembly and results in subunit degradation and complex II deficiency in fetal mice. These defects are exacerbated in young adults with globally impaired metabolic capacity and activation of dynamin-related protein 1, which induces excess mitochondrial fission and mitophagy, thereby causing progressive dilated cardiomyopathy and lethal heart failure in animals. Targeting mitochondria via supplementation with fumarate or inhibiting mitochondrial fission improves mitochondrial dynamics, partially restores cardiac function and prolongs the lifespan of mutant mice. Moreover, the addition of fumarate is found to dramatically improve cardiac function in myocardial infarction mice. These findings reveal a vital role for complex II assembly in the development of dilated cardiomyopathy and provide additional insights into therapeutic interventions for heart diseases.
  18. Cancers (Basel). 2022 Jun 30. pii: 3225. [Epub ahead of print]14(13):
      Neuroblastoma, the most common solid tumor in children, is characterized by amplification of the MYCN proto-oncogene, a high-risk aggressive clinical marker associated with treatment failure. MYCN plays an important role in cell growth, proliferation, metabolism, and chemoresistance. Here, we show for the first time that in neuroblastoma, iron chelator VLX600 inhibits mitochondrial respiration, decreases expression levels of MYCN/LMO1, and induces an efficient cell death regardless of MYCN status in both 2D and 3D culture conditions. Moreover, insufficient induction of autophagy was observed in cells treated with VLX600, which is essential as a protective response in the event of ATP synthesis disruption. Further inhibition of glucose uptake using DRB18, a pan-GLUT (glucose transporter) inhibitor, synergized the effect of VLX600 and no significant cell death was found in immortalized epithelial cells under this combination treatment. Our results demonstrate that inhibition of mitochondrial respiration by iron chelator VLX600 accompanied by autophagy deficiency promotes sensitivity of neuroblastoma cells in a nutrition-restricted microenvironment regardless of MYCN status, indicating that MYCN expression level is an essential clinical marker but might not be a necessary target for the treatment of neuroblastoma which warrants further investigation. VLX600 has been studied in Phase I clinical trials; combining VLX600 with conventional chemotherapy could be an innovative therapeutic strategy for neuroblastoma.
    Keywords:  MYCN; mTOR; mitochondria; neuroblastoma; new therapeutic approach; spheroids
  19. Cancer Res. 2022 Jul 05. 82(13): 2354-2356
      Understanding how carcinogenesis can expose cancers to synthetically lethal vulnerabilities has been an essential underpinning of development of modern anticancer therapeutics. Isocitrate dehydrogenase wild-type (IDHWT) glioblastoma multiforme (GBM), which is known to have upregulated branched-chain amino acid transaminase 1 (BCAT1) expression, has not had treatments developed to the same extent as the IDH mutant counterpart, despite making up the majority of cases. In this issue, Zhang and colleagues utilize a metabolic screen to identify α-ketoglutarate (AKG) as a synthetically lethal treatment in conjunction with BCAT1 inhibition in IDHWT GBM. These treatments synergize in a multipronged approach that limits substrate catabolism and disrupts mitochondrial homeostasis through perturbing the balance of NAD+/NADH, leading to mTORC1 inhibition and a reduction of nucleotide biosynthesis. Based on these results, the authors propose combination treatment targeting branched chain amino acid catabolism as a potential option for patients with IDHWT GBM. See related article by Zhang et al., p. 2388.
  20. Elife. 2022 Jul 08. pii: e76095. [Epub ahead of print]11
      Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.
    Keywords:  S. cerevisiae; cell biology; genetics; genomics
  21. Nat Commun. 2022 Jul 08. 13(1): 3971
      Therapeutic targeting of KRAS-mutant colorectal cancer (CRC) is an unmet need. Here, we show that Proprotein Convertase Subtilisin/Kexin type 9 (PSCK9) promotes APC/KRAS-mutant CRC and is a therapeutic target. Using CRC patient cohorts, isogenic cell lines and transgenic mice, we identify that de novo cholesterol biosynthesis is induced in APC/KRAS mutant CRC, accompanied by increased geranylgeranyl diphosphate (GGPP)─a metabolite necessary for KRAS activation. PCSK9 is the top up-regulated cholesterol-related gene. PCSK9 depletion represses APC/KRAS-mutant CRC cell growth in vitro and in vivo, whereas PCSK9 overexpression induces oncogenesis. Mechanistically, PCSK9 reduces cholesterol uptake but induces cholesterol de novo biosynthesis and GGPP accumulation. GGPP is a pivotal metabolite downstream of PCSK9 by activating KRAS/MEK/ERK signaling. PCSK9 inhibitors suppress growth of APC/KRAS-mutant CRC cells, organoids and xenografts, especially in combination with simvastatin. PCSK9 overexpression predicts poor survival of APC/KRAS-mutant CRC patients. Together, cholesterol homeostasis regulator PCSK9 promotes APC/KRAS-mutant CRC via GGPP-KRAS/MEK/ERK axis and is a therapeutic target.
  22. Nat Commun. 2022 Jul 07. 13(1): 3775
      Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.
  23. Cancers (Basel). 2022 Jun 28. pii: 3154. [Epub ahead of print]14(13):
      The conceptualization of a novel type of cell death, called ferroptosis, opens new avenues for the development of more efficient anti-cancer therapeutics. In this context, a full understanding of the ferroptotic pathways, the players involved, their precise role, and dispensability is prerequisite. Here, we focused on the importance of glutathione (GSH) for ferroptosis prevention in pancreatic ductal adenocarcinoma (PDAC) cells. We genetically deleted a unique, rate-limiting enzyme for GSH biosynthesis, γ-glutamylcysteine ligase (GCL), which plays a key role in tumor cell proliferation and survival. Surprisingly, although glutathione peroxidase 4 (GPx4) has been described as a guardian of ferroptosis, depletion of its substrate (GSH) led preferentially to apoptotic cell death, while classical ferroptotic markers (lipid hydroperoxides) have not been observed. Furthermore, the sensitivity of PDAC cells to the pharmacological/genetic inhibition of GPx4 revealed GSH dispensability in this context. To the best of our knowledge, this is the first time that the complete dissection of the xCT-GSH-GPx4 axis in PDAC cells has been investigated in great detail. Collectively, our results revealed the necessary role of GSH in the overall redox homeostasis of PDAC cells, as well as the dispensability of this redox-active molecule for a specific, antioxidant branch dedicated to ferroptosis prevention.
    Keywords:  GPx4; ferroptosis; glutathine; lipid hydroperoxides; γ-glutamylcysteine ligase
  24. Annu Rev Cell Dev Biol. 2022 Jul 08.
      Mitochondria are traditionally known as the powerhouse of the cell, but their functions extend far beyond energy production. They are vital in cellular and organismal pathways that direct metabolism, stress responses, immunity, and cellular fate. To accomplish these tasks, mitochondria have established networks of both intra- and extracellular communication. Intracellularly, these communication routes comprise direct contacts between mitochondria and other subcellular components as well as indirect vesicle transport of ions, metabolites, and other intracellular messengers. Extracellularly, mitochondria can induce stress responses or other cellular changes that secrete mitochondrial cytokine (mitokine) factors that can travel between tissues as well as respond to immune challenges from extracellular sources. Here we provide a current perspective on the major routes of communication for mitochondrial signaling, including their mechanisms and physiological impact. We also review the major diseases and age-related disorders associated with defects in these signaling pathways. An understanding of how mitochondrial signaling controls cellular homeostasis will bring greater insight into how dysfunctional mitochondria affect health in disease and aging. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see for revised estimates.
  25. Nat Chem Biol. 2022 Jul 04.
      Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.
  26. Cancers (Basel). 2022 Jun 30. pii: 3220. [Epub ahead of print]14(13):
      Biguanides are a family of antidiabetic drugs with documented anticancer properties in preclinical and clinical settings. Despite intensive investigation, how they exert their therapeutic effects is still debated. Many studies support the hypothesis that biguanides inhibit mitochondrial complex I, inducing energy stress and activating compensatory responses mediated by energy sensors. However, a major concern related to this "complex" model is that the therapeutic concentrations of biguanides found in the blood and tissues are much lower than the doses required to inhibit complex I, suggesting the involvement of additional mechanisms. This comprehensive review illustrates the current knowledge of pharmacokinetics, receptors, sensors, intracellular alterations, and the mechanism of action of biguanides in diabetes and cancer. The conditions of usage and variables affecting the response to these drugs, the effect on the immune system and microbiota, as well as the results from the most relevant clinical trials in cancer are also discussed.
    Keywords:  biguanides; cancer; complex I; metabolism; metformin; redox
  27. Aging (Albany NY). 2022 Jul 07. 14(undefined):
      One of the biological features of cancer cells is their aerobic glycolysis by extensive glucose fermentation to harvest energy, so called Warburg effect. Melanoma is one of the most aggressive human cancers with poor prognosis and high mortality for its high metastatic ability. During the metastatic process, the metastatic tumor cells should survive under detachment stress. However, whether the detachment stress could affect the tumor phenotype is worthy to investigate. We had established the cell model of human melanoma cells under detachment stress, which mimicked circulating melanoma. It had been demonstrated that the detachment stress altered melanoma cell activities, malignancy, and drug sensitivity. In this study, we found that adherent melanoma cells were more sensitive to glucose depletion. Gene expression profiling altered expressions of transporters associated with glucose metabolism. In addition, detachment stress reduced lactate secretion owing to the reduced MCT4 and GLUT1 expressions, the altered glycolytic and respiratory capacities, and the increased superoxide production. Detachment stress also increases the sensitivity of melanoma cells toward the blockade of electron transport chains. Investigation of the change in glucose metabolism of melanoma cells under detachment stress would be critical to provide a novel molecular mechanism to develop potential therapeutics.
    Keywords:  anti-Warburg; detachment stress; electron transport chain; glycolysis; melanoma
  28. Nutrients. 2022 Jul 01. pii: 2752. [Epub ahead of print]14(13):
      Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD+), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD+ precursors influence intracellular NAD+ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD+ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD+ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD+ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD+ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD+ precursor in mammalian cells and how different NAD+ precursors can interact and influence each other when co-administered.
    Keywords:  NAD+; dihydronicotinamide riboside; dihydronicotinic acid riboside; nicotinamide; nicotinamide riboside; nicotinic acid; nicotinic acid riboside; vitamin B3
  29. Discov Oncol. 2022 Jul 08. 13(1): 60
      Metabolic reprogramming (MR) influences progression of chronic myeloid leukaemia (CML) to blast crisis (BC), but metabolic programs may change transiently in a second dimension (metabolic plasticity, MP), driven by environments as hypoxia, affecting cytotoxic potency (CPot) of drugs targeting mitochondria or mitochondria-related cell stress responses (MRCSR) such as mitophagy and mitochondrial biogenesis. We assessed mitochondrial membrane potential (MMP), mitochondrial mass (MM), apoptosis, glucose uptake (GU), and CPot of arsenic trioxide (ATO), CCCP, valproic acid (VPA), vincristine (VCR), Mdivi1, and dichloroacetic acid (DCA) in CML BC cells K562 (BC-K562) under hypoxia through flow cytometry, and gene expression from GEO database. About 60% of untreated cells were killed after 72 h under hypoxia, but paradoxically, all drugs but ATO rescued cells and increased survival rates to almost 90%. Blocking mitophagy either with VCR or Mdivi1, or increasing mitochondrial biogenesis with VPA enhanced cell-survival with increased MM. DCA increased MM and rescued cells in spite of its role in activating pyruvate dehydrogenase and Krebs cycle. Cells rescued by DCA, VPA and CCCP showed decreased GU. ATO showed equal CPot in hypoxia and normoxia. MP was evidenced by differential expression of genes (DEG) under hypoxia related to Krebs cycle, lipid synthesis, cholesterol homeostasis, mitophagy, and mitochondrial biogenesis (GSE144527). A 25-gene MP-signature of BC-K562 cells under hypoxia identified BC cases among 113 transcriptomes from CML patients (GSE4170). We concluded that hypoxic environment drove a MP change evidenced by DEG that was reflected in a paradoxical pro-survival, instead of cytotoxic, effect of drugs targeting mitochondria and MRCSR.
    Keywords:  Arsenic Trioxide; Dichloroacetate; Gene expression profiling; Glucose uptake; Metabolic reprogramming; Valproic acid
  30. Front Microbiol. 2022 ;13 864006
      During respiration, adenosine triphosphate (ATP) synthases harness the electrochemical proton motive force (PMF) generated by the electron transport chain (ETC) to synthesize ATP. These macromolecular machines operate by a remarkable rotary catalytic mechanism that couples transmembrane proton translocation to rotation of a rotor subcomplex, and rotation to ATP synthesis. Initially, x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cross-linking were the only ways to gain insights into the three-dimensional (3D) structures of ATP synthases and, in particular, provided ground-breaking insights into the soluble parts of the complex that explained the catalytic mechanism by which rotation is coupled to ATP synthesis. In contrast, early electron microscopy was limited to studying the overall shape of the assembly. However, advances in electron cryomicroscopy (cryoEM) have allowed determination of high-resolution structures, including the membrane regions of ATP synthases. These studies revealed the high-resolution structures of the remaining ATP synthase subunits and showed how these subunits work together in the intact macromolecular machine. CryoEM continues to uncover the diversity of ATP synthase structures across species and has begun to show how ATP synthases can be targeted by therapies to treat human diseases.
    Keywords:  ATP synthase; bioenergetics; cryoEM; membrane; protein; structure
  31. Front Oncol. 2022 ;12 887257
      Branched-chain amino acids (BCAAs) are the three essential amino acids including leucine, isoleucine, and valine. BCAA metabolism has been linked with the development of a variety of tumors. However, the impact of dietary BCAA intake on breast tumor progression and metastasis remains to be fully explored. Here, we unexpectedly find that the elevated BCAA, either in the genetic model or via increasing dietary intake in mice, suppresses the tumor growth and lung metastasis of breast cancer. The survival analysis shows that BCAA catabolic gene expression is strongly associated with long-term oncological outcomes in patients with breast cancer. In Pp2cm knockout mice in which BCAAs accumulate due to the genetic defect of BCAA catabolism, the breast tumor growth is suppressed. Interestingly, while the cell proliferation and tumor vasculature remain unaffected, more cell death occurs in the tumor in Pp2cm knockout mice, accompanied with increased natural killer (NK) cells. Importantly, increasing BCAA dietary intake suppresses breast tumor growth in mice. On the other hand, there are fewer lung metastases from primary breast tumor in Pp2cm knockout mice and the high BCAA diet-fed mice, suggesting high BCAA also suppresses the lung metastasis of breast cancer. Furthermore, low BCAA diet promotes lung colonization of breast cancer cells in tail vein model. The migration and invasion abilities of breast cancer cells are impaired by high concentration of BCAA in culture medium. The suppressed tumor metastasis and cell migration/invasion abilities by elevated BCAA are accompanied with reduced N-cadherin expression. Together, these data show high BCAA suppresses both tumor growth and metastasis of breast cancer, demonstrating the potential benefits of increasing BCAA dietary intake in the treatment of breast cancer.
    Keywords:  N-Cadherin; NK cell; branched-chain amino acid; breast cancer; metastasis
  32. Int J Mol Sci. 2022 Jun 21. pii: 6887. [Epub ahead of print]23(13):
      Gold nanoparticles (AuNP) can increase the efficacy of radiation therapy by sensitising tumor cells to radiation damage. When used in combination with radiation, AuNPs enhance the rate of cell killing; hence, they may be of great value in radiotherapy. This study assessed the effects of radiation and AuNPs on mitochondrial reactive oxygen species (ROS) generation in cancer cells as an adjunct therapeutic target in addition to the DNA of the cell. Mitochondria are considered one of the primary sources of cellular ROS. High levels of ROS can result in an intracellular state of oxidative stress, leading to permanent cell damage. In this study, human melanoma and prostate cancer cell lines, with and without AuNPs, were irradiated with 6-Megavolt X-rays at doses of 0-8 Gy. Indicators of mitochondrial stress were quantified using two techniques, and were found to be significantly increased by the inclusion of AuNPs in both cell lines. Radiobiological damage to mitochondria was quantified via increased ROS activity. The ROS production by mitochondria in cells was enhanced by the inclusion of AuNPs, peaking at ~4 Gy and then decreasing at higher doses. This increased mitochondrial stress may lead to more effectively kill of AuNP-treated cells, further enhancing the applicability of functionally-guided nanoparticles.
    Keywords:  cancer cell lines; gold nanoparticles (AuNP); mitochondria; radiation; reactive oxygen species
  33. Nat Commun. 2022 Jul 06. 13(1): 3882
      Mitochondrial dynamics can regulate Major Histocompatibility Complex (MHC)-I antigen expression by cancer cells and their immunogenicity in mice and in patients with malignancies. A crucial role in the mitochondrial fragmentation connection with immunogenicity is played by the IRE1α-XBP-1s axis. XBP-1s is a transcription factor for aminopeptidase TPP2, which inhibits MHC-I complex cell surface expression likely by degrading tumor antigen peptides. Mitochondrial fission inhibition with Mdivi-1 upregulates MHC-I expression on cancer cells and enhances the efficacy of adoptive T cell therapy in patient-derived tumor models. Therefore mitochondrial fission inhibition might provide an approach to enhance the efficacy of T cell-based immunotherapy.
  34. Integr Biol (Camb). 2022 Jul 02. pii: zyac008. [Epub ahead of print]
      Cancer is a highly fatal disease without effective early-stage diagnosis and proper treatment. Along with the oncoproteins and oncometabolites, several organelles from cancerous cells are also emerging as potential biomarkers. Mitochondria isolated from cancer cells are one such biomarker candidates. Cancerous mitochondria exhibit different profiles compared with normal ones in morphology, genomic, transcriptomic, proteomic and metabolic landscape. Here, the possibilities of exploring such characteristics as potential biomarkers through single-cell omics and Artificial Intelligence (AI) are discussed. Furthermore, the prospects of exploiting the biomarker-based diagnosis and its futuristic utilization through circulatory tumor cell technology are analyzed. A successful alliance of circulatory tumor cell isolation protocols and a single-cell omics platform can emerge as a next-generation diagnosis and personalized treatment procedure.
    Keywords:  cancer biomarker; circulatory tumor cell; mitochondria; mtDNA; single-cell omics
  35. Anticancer Res. 2022 Jul;42(7): 3313-3324
      BACKGROUND/AIM: Resistance to chemotherapy is a major obstacle for patients with unresectable colorectal cancer (CRC); however, the factors that induce chemoresistance have not been elucidated. Lipid composition influences neoplastic behaviour. Therefore, this study examined whether lipid composition affects sensitivity to chemotherapeutic agents in CRC.MATERIALS AND METHODS: We performed a lipidomic analysis of a CRC xenograft-derived spheroid model to identify potential relationships between the lipid profile and chemoresistance to 5-fluorouracil (5-FU). Genetic and pharmacological modulation of lipid synthesis were also used in the HCT-116 and DLD-1 CRC cell lines to further characterize resistance to 5-FU.
    RESULTS: Our lipidomic profiling revealed that phospholipids with saturated fatty acids (SFAs) were more abundant in 5-FU-resistant spheroids. The importance of phospholipids containing SFA in chemoresistance was confirmed by showing that in HCT-116 and DLD-1 cells, genetic or pharmacological inactivation of stearoyl-CoA desaturase-1, a key enzyme that converts SFAs to monounsaturated fatty acids, increased the proportion of SFAs in membranous phospholipids and reduced cell membrane fluidity, and this ultimately resulted in resistance to 5-FU.
    CONCLUSION: These data suggest that the saturated to monounsaturated fatty acid ratio in cellular membranous phospholipids affects sensitivity to chemotherapeutic agents.
    Keywords:  Saturated fatty acid; chemoresistance; liquid chromatography mass spectrometry; membrane fluidity; stearoyl-CoA desaturase
  36. Cancer Lett. 2022 Jul 05. pii: S0304-3835(22)00284-1. [Epub ahead of print] 215800
      Cancer cells thrive when embedded in a fine-tuned cellular and extracellular environment or tumour microenvironment (TME). There is a general understanding of a co-evolution between cancer cells and their surrounding TME, pointing at a functional connection between cancer cells characteristics and the perturbations induced in their surrounding tissue. However, it has never been formally proven whether this functional connection needs to be set from the start or if aggressive cancer cells always dominate their microenvironmental any point in time. This would require a dedicated experimental setting where malignant cells are challenged to grow in a different TME from the one they would naturally create. Here we generated an experimental setting where we transiently perturb the secretory profile of aggressive breast cancer cells without affecting their intrinsic growth ability, which led to the initial establishment of an atypical TME. Interestingly, even if initially tumours are formed, this atypical TME evolves to impair long term in vivo cancer growth. Using a combination of in vivo transcriptomics, protein arrays and in vitro co-cultures, we found that the atypical TME culminates in the infiltration of macrophages with STAT1high activity. These macrophages show strong anti-tumoural functions which reduce long-term tumour growth, despite lacking canonical M1 markers. Importantly, gene signatures of the mesenchymal compartment of the TME, as well as the anti-tumoural macrophages, show striking prognostic power that correlates with less aggressive human breast cancers.
  37. Biochim Biophys Acta Bioenerg. 2022 Jun 30. pii: S0005-2728(22)00056-1. [Epub ahead of print]1863(7): 148587
      Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.
    Keywords:  Cardiolipin; Linoleic acid; Membrane; Mitochondria; Yeast
  38. Biochim Biophys Acta Mol Cell Res. 2022 Jul 04. pii: S0167-4889(22)00115-X. [Epub ahead of print]1869(10): 119323
      Thioredoxin reductase (TrxR) is a pivotal regulator of redox homeostasis, while dysregulation of redox homeostasis is a hallmark for cancer cells. Thus, there is considerable potential to inhibit the aberrantly upregulated TrxR in cancer cells to discover selective cancer therapeutic agents. Nevertheless, the structural types of TrxR inhibitors presented currently are still relatively limited. We herein report that PACMA 31, previously reported to inhibit protein disulfide isomerase (PDI), is a potent TrxR inhibitor. PACMA 31 possesses a pharmacophore scaffold that is structurally different from the announced TrxR inhibitors and exhibits effective cytotoxicity against cervical cancer cells. Our results reveal that PACMA 31 selectively inhibits TrxR over the related glutathione reductase (GR) and in the presence of reduced glutathione (GSH). Further studies with mutant enzyme and molecular docking suggest that the propynamide fragment of PACMA 31 interacts covalently with the selenocysteine residue of TrxR. Moreover, PACMA 31 effectively and selectively curbs TrxR activity in cells and further stimulates the production of reactive oxygen species (ROS) at low micromolar concentrations, which in turn triggers the accumulation of oxidized thioredoxin (Trx) and GSSG in cells. Follow-up studies demonstrate that PACMA 31 targets TrxR in cells to induce oxidative stress-mediated cancer cell apoptosis. Our results provide a new structural type of TrxR inhibitor that may serve as a useful probe for investigating the biology of TrxR-implicated pathways, and uncover a new target of PACMA 31 that contributes to it becoming a candidate for cancer treatment.
    Keywords:  Apoptosis; Oxidative stress; PACMA 31; Reactive oxygen species; Thioredoxin; Thioredoxin reductase
  39. Blood Adv. 2022 Jul 06. pii: bloodadvances.2022007250. [Epub ahead of print]
      Genome-wide CRISPR screens have been extremely useful to identify therapeutic targets in diverse cancers, by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in two in vivo murine models of AML driven by the KMT2A/MLLT3 fusion or by the constitutive co-expression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the MHC class I complex, Cd47, complement receptor Cr1l, and the beta-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as an in vivo-specific dependency with high prognostic relevance. Overall, we demonstrate an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.
  40. Cancer Res. 2022 Jul 06. pii: can.21.2908. [Epub ahead of print]
      Many advanced therapeutics possess cytostatic properties that suppress cancer cell growth without directly inducing death. Treatment-induced cytostatic cancer cells can persist and constitute a reservoir from which recurrent growth and resistant clones can develop. Current management approaches primarily comprise maintenance and monitoring because strategies for targeting non-proliferating cancer cells have been elusive. Here, we utilized targeted therapy paradigms and engineered cytostatic states to explore therapeutic opportunities for depleting treatment-mediated cytostatic cancer cells. Sustained oncogenic AKT signaling was common, while non-essential, in treatment-mediated cytostatic cancer cells harboring PI3K-pathway mutations, which are associated with cancer recurrence. Engineering oncogenic signals in quiescent mammary organotypic models showed that sustained, aberrant activation of AKT sensitized cytostatic epithelial cells to proteasome inhibition. Mechanistically, sustained AKT signaling altered cytostatic state homeostasis and promoted an oxidative and proteotoxic environment, which imposed an increased proteasome dependency for maintaining cell viability. Under cytostatic conditions, inhibition of the proteasome selectively induced apoptosis in the population with aberrant AKT activation compared to normal cells. Therapeutically exploiting this AKT-driven proteasome vulnerability was effective in depleting treatment-mediated cytostatic cancer cells independent of breast cancer subtype, epithelial origin, and cytostatic agent. Moreover, transient targeting during cytostatic treatment conditions was sufficient to reduce recurrent tumor growth in spheroid and mouse models. This work identified an AKT-driven proteasome-vulnerability that enables depletion of persistent cytostatic cancer cells harboring PTEN/PI3K pathway mutations, revealing a viable strategy for targeting non-proliferating persistent cancer cell populations before drug resistance emerges.
  41. Front Microbiol. 2022 ;13 872565
      In FOF1 ATP synthase, driven by the proton motive force across the membrane, the FO motor rotates the central rotor and induces conformational changes in the F1 motor, resulting in ATP synthesis. Recently, many near-atomic resolution structural models have been obtained using cryo-electron microscopy. Despite high resolution, however, static information alone cannot elucidate how and where the protons pass through the FO and how proton passage is coupled to FO rotation. Here, we review theoretical and computational studies based on FO structure models. All-atom molecular dynamics (MD) simulations elucidated changes in the protonation/deprotonation of glutamate-the protein-carrier residue-during rotation and revealed the protonation states that form the "water wire" required for long-range proton hopping. Coarse-grained MD simulations unveiled a free energy surface based on the protonation state and rotational angle of the rotor. Hybrid Monte Carlo and MD simulations showed how proton transfer is coupled to rotation.
    Keywords:  FO motor; FOF1 ATP synthases; Monte Carlo simulations; coarse-grained model; molecular dynamics simulations
  42. J Biol Chem. 2022 Jun 29. pii: S0021-9258(22)00658-5. [Epub ahead of print] 102216
      Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cell-, membrane- and vesicle-fractions suggest that this proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. Using the His-tag, the enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7-8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.
    Keywords:  Thermoanaerobacter kivui; acetogenic metabolism; energy-converting hydrogenase (Ech); extremophile; proteoliposomes; proton translocation