ABSTRACT
Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse. SIGNIFICANCE: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.
Subject(s)
Brain Neoplasms , Cerebellar Neoplasms , Medulloblastoma , Animals , Humans , Mice , Cerebellar Neoplasms/pathology , Medulloblastoma/pathology , Neoplasm Recurrence, Local/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Transcription Factors/metabolismABSTRACT
Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
Subject(s)
Mitochondria/genetics , Mitophagy/genetics , RNA-Binding Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Adaptor Proteins, Signal Transducing/genetics , Autophagy , HeLa Cells , Humans , Mitochondrial Proteins/genetics , Polyubiquitin/genetics , Sequestosome-1 Protein/genetics , Ubiquitination/geneticsABSTRACT
SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Breast/pathology , CDC2 Protein Kinase/metabolism , Cyclin-Dependent Kinase 2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Amino Acid Sequence , Breast/metabolism , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Phosphorylation , Prognosis , Promoter Regions, Genetic , Proteolysis , SKP Cullin F-Box Protein Ligases/analysis , SKP Cullin F-Box Protein Ligases/genetics , Signal Transduction , Survival Analysis , Transcriptional Activation , UbiquitinationABSTRACT
TRIM16 exhibits tumour suppressor functions by interacting with cytoplasmic vimentin and nuclear E2F1 proteins in neuroblastoma and squamous cell carcinoma cells, reducing cell migration and replication. Reduced TRIM16 expression in a range of human primary malignant tissues correlates with increased malignant potential. TRIM16 also induces apoptosis in breast and lung cancer cells, by unknown mechanisms. Here we show that overexpression of TRIM16 induces apoptosis in human breast cancer (MCF7) and neuroblastoma (BE(2)-C) cells, but not in non-malignant HEK293 cells. TRIM16 increased procaspase-2 protein levels in MCF7 and induced caspase-2 activity in both MCF7 and BE(2)-C cells. We show that TRIM16 and caspase-2 proteins directly interact in both MCF7 and BE(2)-C cells and co-localise in MCF7 cells. Most importantly, the induction of caspase-2 activity is required for TRIM16 to initiate apoptosis. Our data suggest a novel mechanism by which TRIM16 can promote apoptosis by directly modulating caspase-2 activity.
Subject(s)
Apoptosis/genetics , Caspase 2/genetics , Cysteine Endopeptidases/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Transcription Factors/genetics , Caspase 2/metabolism , Cell Line, Tumor , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , HEK293 Cells , Humans , Membrane Potential, Mitochondrial/genetics , Mitochondria/genetics , Mitochondria/metabolism , Organ Specificity , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Tripartite Motif Proteins , Ubiquitin-Protein LigasesABSTRACT
Selenium is a trace element essential for normal physiological processes. Organic selenium-containing amino acids, such as selenocysteine (Sec) / selenocystine and selenomethionine (SeMet, the major dietary form), can provide antioxidant benefits by acting both as direct antioxidants as well as a source of selenium for synthesis of selenium-dependent antioxidant and repair proteins (e.g., glutathione peroxidases, thioredoxin reductases, methionine sulfoxide reductases). The direct antioxidant actions of these amino acids arise from the nucleophilic properties of the ionized selenol (RSe(-), which predominates over the neutral form at physiological pH values) and the ease of oxidation of Sec and SeMet. This results in higher rate constants for reaction with multiple oxidants, than for the corresponding thiols/thioethers. Furthermore, the resulting oxidation products are more readily and rapidly reversed by both enzyme and nonenzymatic reactions. The antioxidant effects of these seleno species can therefore be catalytic. Seleno amino acids may also chelate redox-active metal ions. The presence of Sec in the catalytic site of selenium-dependent antioxidant enzymes enhances the kinetic properties and broadens the catalytic activity of antioxidant enzymes against biological oxidants when compared with sulfur-containing species. However, while normal physiological selenium levels afford protection, when compared with deficiency, excessive selenium may induce damage and adverse effects, with this being manifest, for example, as an increased incidence of type 2 diabetes. Further studies examining the availability of redox-active selenium species and their mechanisms and kinetics of action are therefore of critical importance in the potential development of seleno species as a therapeutic strategy.
Subject(s)
Amino Acids/pharmacology , Antioxidants/pharmacology , Selenium/pharmacology , Animals , Humans , Oxidation-ReductionABSTRACT
OBJECTIVE: People with diabetes have an elevated risk of atherosclerosis. The accumulation of lipid within macrophage cells in the artery wall is believed to arise via the uptake and subsequent processing of modified low-density lipoproteins (LDL) via the endo-lysosomal system. In this study the effects of prolonged exposure to elevated glucose upon macrophage lysosomal function was examined to determine whether this contributes to modulated protein catabolism. METHODS: Human monocytes were isolated from white-cell concentrates and differentiated, in vitro, into monocyte-derived macrophages over 11 days in medium containing 5-30 mmol/L glucose. Murine macrophage-like J774A.1 cells were incubated similarly. Lysosomal cathepsin (B, D, L and S) and acid lipase activities were assessed using fluorogenic substrates; cathepsin protein levels were examined by Western blotting. Lysosomal numbers were examined using the lysomotropic fluorescent dye LysoTracker DND-99, measurement of aryl sulfatase activity, and quantification of lysosome-associated membrane glycoprotein-1 (LAMP-1) by Western blotting. RESULTS: Exposure to elevated glucose, but not mannitol, resulted in a concentration-dependent decrease in the activity, and to a lesser extent protein levels, of four lysosomal cathepsins. Acid lipase activity was also significantly reduced. Arysulfatase activity, LAMP-1 levels and lysosomal numbers were also decreased at the highest glucose concentrations, though to a lesser extent. CONCLUSION: Long term exposure of human and murine macrophage cells to elevated glucose levels result in a depression of lysosomal proteolytic and lipase activities. This may result in decreased clearance and cellular accumulation of (lipo)proteins and contribute to the accumulation of modified proteins and lipids in diabetes-associated atherosclerosis.
Subject(s)
Atherosclerosis/etiology , Diabetic Angiopathies/etiology , Glucose/metabolism , Lysosomes/metabolism , Macrophages/metabolism , Animals , Arylsulfatases/metabolism , Atherosclerosis/metabolism , Blotting, Western , Cathepsins/metabolism , Cell Line , Diabetic Angiopathies/metabolism , Down-Regulation , Humans , Lysosomal Membrane Proteins/metabolism , Mice , Microscopy, Fluorescence , Sterol Esterase/metabolism , Time FactorsABSTRACT
Photo-induced damage to proteins occurs via multiple pathways. Direct damage induced by UVB (λ 280-320 nm) and UVA radiation (λ 320-400 nm) is limited to a small number of amino acid residues, principally tryptophan (Trp), tyrosine (Tyr), histidine (His) and disulfide (cystine) residues, with this occurring via both excited state species and radicals. Indirect protein damage can occur via singlet oxygen ((1)O(2)(1)Δ(g)), with this resulting in damage to Trp, Tyr, His, cystine, cysteine (Cys) and methionine (Met) residues. Although initial damage is limited to these residues multiple secondary processes, that occur both during and after radiation exposure, can result in damage to other intra- and inter-molecular sites. Secondary damage can arise via radicals (e.g. Trp, Tyr and Cys radicals), from reactive intermediates generated by (1)O(2) (e.g. Trp, Tyr and His peroxides) and via molecular reactions of photo-products (e.g. reactive carbonyls). These processes can result in protein fragmentation, aggregation, altered physical and chemical properties (e.g. hydrophobicity and charge) and modulated biological turnover. Accumulating evidence implicates these events in cellular and tissue dysfunction (e.g. apoptosis, necrosis and altered cell signaling), and multiple human pathologies.
Subject(s)
Photochemistry , Proteins/chemistry , Disease , Humans , Oxidation-ReductionABSTRACT
Reaction of radicals and singlet oxygen ((1)O(2)) with proteins results in both direct damage and the formation of long-lived reactive hydroperoxides. Elevated levels of protein hydroperoxide-derived products have been detected in multiple human pathologies, suggesting that these secondary oxidants contribute to tissue damage. Previous studies have provided evidence for protein hydroperoxide-mediated inhibition of thiol-dependent enzymes and modulation of signaling processes in isolated systems. In this study (1)O(2) and hydroperoxides have been generated in J774A.1 macrophage-like cells using visible light and the photosensitizer rose bengal, with the consequences of oxidant formation examined both immediately and after subsequent (dark-phase) incubation. Significant losses of GSH (≤50%), total thiols (≤20%), and activity of thiol-dependent proteins (GAPDH, thioredoxin, protein tyrosine phosphatases, creatine kinase, and cathepsins B and L; 10-50% inhibition) were detected after 1 or 2 min photo-oxidation. Non-thiol-dependent enzymes were not affected. In contrast, NADPH levels increased, together with the activity of glutathione reductase, glutathione peroxidase, and thioredoxin reductase; these increases may be components of a rapid global cytoprotective cellular response to stress. Neither oxidized thioredoxin nor radical-mediated protein oxidation products were detected at significant levels. Further decreases in thiol levels and enzyme activity occurred during dark-phase incubation, with this accompanied by decreased cell viability. These secondary events are ascribed to the reactions of long-lived hydroperoxides, generated by (1)O(2)-mediated reactions. Overall, this study provides novel insights into early cellular responses to photo-oxidative damage and indicates that long-lived hydroperoxides can play a significant role in cellular damage.
Subject(s)
Hydrogen Peroxide/metabolism , Macrophages/metabolism , Oxidants/metabolism , Photochemical Processes , Animals , Antioxidants/metabolism , Cell Survival/drug effects , Cells, Cultured , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Humans , Macrophages/drug effects , Mice , Proteins/metabolism , Rose Bengal/metabolism , Singlet Oxygen/metabolism , Sulfhydryl Compounds/metabolism , Thioredoxins/metabolismABSTRACT
Hydroperoxides are major products of the reactions of radicals and singlet oxygen with amino acids, peptides, and proteins. These species can generate radicals in the presence of metal ions and oxidize thiols via nonradical reactions, but the effects of these materials on cells are poorly understood. In this study the exposure of murine macrophage-like cells to preformed peptide or protein hydroperoxides is shown to result in hydroperoxide consumption and cellular thiol oxidation; these effects precede loss of cell viability. N-acetyltryptophan methyl ester hydroperoxides, but not the decomposed species, decreased total cellular thiols and GSH, with the latter occurring more rapidly. Time-dependent inhibition of lysosomal cathepsins B and L was also observed, together with diminished caspase 3/7 activity. A number of other cytosolic thiol- and non-thiol-dependent enzymes were not affected significantly. Hydroperoxides formed on BSA did not deplete total thiols or GSH within cells, although such reactions are rapid in model systems. In contrast, selective inhibition of cathepsins B and L (but not cathepsin D or arylsulfatase) of the endosomal-lysosomal system was detected, consistent with localization within these compartments. Decomposed BSA hydroperoxides did not induce these effects, indicating a requirement for the hydroperoxide group. The differences between these hydroperoxides are ascribed to their mechanisms of penetration into cells. Overall these studies provide valuable data on the initial cellular events arising from exposure to exogenous protein and amino acid peroxides and indicate that cellular thiols are a major target. This selective oxidation may modulate cellular redox balance and subsequent cell behavior.
Subject(s)
Macrophages/metabolism , Peptides/chemistry , Peroxides/metabolism , Peroxides/pharmacology , Proteins/chemistry , Serum Albumin, Bovine/metabolism , Tryptophan/analogs & derivatives , Animals , Apoptosis/drug effects , Cathepsin B/antagonists & inhibitors , Cathepsin L/antagonists & inhibitors , Cell Line , Cell Survival/drug effects , Glutathione/metabolism , Glutathione Disulfide/metabolism , Macrophages/drug effects , Mice , Serum Albumin, Bovine/pharmacology , Sulfhydryl Compounds/metabolism , Tryptophan/metabolism , Tryptophan/pharmacologyABSTRACT
Maintenance of an adequate supply of cholesterol is important for neuronal function, whereas excess cholesterol promotes amyloid precursor protein (APP) cleavage generating toxic amyloid-beta (Abeta) peptides. To gain insights into the pathways that regulate neuronal cholesterol level, we investigated the potential for reconstituted apolipoprotein E (apoE) discs, resembling nascent lipoprotein complexes in the central nervous system, to stimulate neuronal [3H]cholesterol efflux. ApoE discs potently accelerated cholesterol efflux from primary human neurons and cell lines. The process was saturable (17.5 microg of apoE/ml) and was not influenced by APOE genotype. High performance liquid chromatography analysis of cholesterol and cholesterol metabolites effluxed from neurons indicated that <25% of the released cholesterol was modified to polar products (e.g. 24-hydroxycholesterol) that diffuse from neuronal membranes. Thus, most cholesterol (approximately 75%) appeared to be effluxed from neurons in a native state via a transporter pathway. ATP-binding cassette transporters ABCA1, ABCA2, and ABCG1 were detected in neurons and neuroblastoma cell lines and expression of these cDNAs revealed that ABCA1 and ABCG1 stimulated cholesterol efflux to apoE discs. In addition, ABCA1 and ABCG1 expression in Chinese hamster ovary cells that stably express human APP significantly reduced Abeta generation, whereas ABCA2 did not modulate either cholesterol efflux or Abeta generation. These data indicate that ABCA1 and ABCG1 play a significant role in the regulation of neuronal cholesterol efflux to apoE discs and in suppression of APP processing to generate Abeta peptides.