RESUMO
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Leiomiomatose , Neoplasias Cutâneas , Neoplasias Uterinas , Feminino , Humanos , Carcinoma de Células Renais/genética , Leiomiomatose/genética , Leiomiomatose/patologia , Fumarato Hidratase/genética , Fumarato Hidratase/análise , Neoplasias Renais/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Mutação , RNA Mensageiro/genéticaRESUMO
Biologically important 2-hydroxy carboxylates such as lactate, malate, and 2-hydroxyglutarate exist in two enantiomeric forms that cannot be distinguished under achiral conditions. The D and L (or R, S) enantiomers have different biological origins and functions, and therefore, there is a need for a simple method for resolving, identifying, and quantifying these enantiomers. We have adapted and improved a chiral derivatization technique for nuclear magnetic resonance (NMR), which needs no chromatography for enantiomer resolution, with greater than 90% overall recovery. This method was developed for 2-hydroxyglutarate (2HG) to produce diastereomers resolvable by column chromatography. We have applied the method to lactate, malate, and 2HG. The limit of quantification was determined to be about 1 nmol for 2HG with coefficients of variation of less than 5%. We also demonstrated the method on an extract of a renal carcinoma bearing an isocitrate dehydrogenase-2 (IDH2) variant that produces copious quantities of 2HG and showed that it is the D enantiomer that was exclusively produced. We also demonstrated in the same experiment that the lactate produced in the same sample was the L enantiomer.
Assuntos
Neoplasias Renais , Malatos , Humanos , Hidroxiácidos , Isocitrato Desidrogenase , Lactatos , Espectroscopia de Ressonância MagnéticaRESUMO
An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.
Assuntos
Cisteína/metabolismo , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Neoplasias Renais/metabolismo , Leiomiomatose/metabolismo , Síndromes Neoplásicas Hereditárias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Uterinas/metabolismo , Linhagem Celular Tumoral , Cisteína/genética , Fumarato Hidratase/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Leiomiomatose/genética , Leiomiomatose/patologia , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologiaRESUMO
Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
Assuntos
Fumaratos/metabolismo , Leiomiomatose/metabolismo , Síndromes Neoplásicas Hereditárias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Uterinas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Cisteína , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Leiomiomatose/genética , Modelos Biológicos , Síndromes Neoplásicas Hereditárias/genética , Proteômica , Transdução de Sinais , Neoplasias Cutâneas/genética , Espectrometria de Massas em Tandem/métodos , Neoplasias Uterinas/genéticaRESUMO
Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a â¼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.
Assuntos
Reprogramação Celular , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Enxofre/metabolismo , Aconitato Hidratase/metabolismo , Metabolismo Energético , Células HEK293 , Humanos , Redes e Vias MetabólicasRESUMO
Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.
Assuntos
Fluorescência , Fumaratos/análise , Fumaratos/efeitos da radiação , Linhagem Celular , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/efeitos da radiação , Fumaratos/metabolismo , Humanos , Microscopia Confocal , Estrutura Molecular , Imagem Óptica , Tetrazóis/químicaRESUMO
OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.
Assuntos
Autofagia/genética , Lisossomos/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Canais de Potássio/deficiência , Idade de Início , Pré-Escolar , Feminino , Humanos , Lactente , Lisossomos/patologia , Masculino , Mutação , Linhagem , Canais de Potássio/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
ISCU myopathy is an inherited disease that primarily affects individuals of northern Swedish descent who share a single point mutation in the fourth intron of the ISCU gene. The current study shows correction of specific phenotypes associated with disease following treatment with an antisense oligonucleotide (ASO) targeted to the site of the mutation. We have shown that ASO treatment diminished aberrant splicing and increased ISCU protein levels in both patient fibroblasts and patient myotubes in a concentration dependent fashion. Upon ASO treatment, levels of SDHB in patient myotubular cell lines increased to levels observed in control myotubular cell lines. Additionally, we have shown that both patient fibroblast and myotubular cell lines displayed an increase in complex II activity with a concomitant decrease in succinate levels in patient myotubular cell lines after ASO treatment. Mitochondrial and cytosolic aconitase activities increased significantly following ASO treatment in patient myotubes. The current study suggests that ASO treatment may serve as a viable approach to correcting ISCU myopathy in patients.
Assuntos
Acidose Láctica/congênito , Proteínas Ferro-Enxofre/genética , Doenças Musculares/congênito , Oligonucleotídeos Antissenso/genética , Succinato Desidrogenase/genética , Acidose Láctica/genética , Acidose Láctica/patologia , Acidose Láctica/terapia , Feminino , Humanos , Íntrons/genética , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Doenças Musculares/genética , Doenças Musculares/patologia , Doenças Musculares/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Fenótipo , Mutação Puntual , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Succinato Desidrogenase/biossínteseRESUMO
Numerous diverse biological pathways are dysregulated in the epileptic focus. Which of these pathways are most critical in producing the biological abnormalities that lead to epilepsy? Answering this question is key to identifying the primary causes of epilepsy and for discovering new therapeutic strategies with greater efficacy than currently available antiepileptics (AEDs). We have performed the largest genome-wide transcriptomic analysis to date comparing epileptic with normal human hippocampi. We have identified 118 differentially expressed and, for the first time, differentially connected pathways in the epileptic focus. Using network mapping techniques, we have shown that these dysregulated pathways, though seemingly disparate, form a coherent interconnected central network. Using closeness centrality analysis, we have identified that the most influential hub pathways in this network are signalling through G protein-coupled receptors, in particular opioid receptors, and their downstream effectors PKA/CREB and DAG/IP3. Next, we have objectively demonstrated that genetic association of gene sets in independent genome-wide association studies (GWASs) can be used to identify causally relevant gene sets: we show that proven causal epilepsy genes, which cause familial Mendelian epilepsy syndromes, are associated in published sporadic epilepsy GWAS results. Using the same technique, we have shown that central pathways identified (opioid receptor and PKA/CREB and DAG/IP3 signalling pathways) are genetically associated with focal epilepsy and, hence, likely causal. Published functional studies in animal models provide evidence of a role for these pathways in epilepsy. Our work shows that these pathways play a central role in human focal epilepsy and that they are important currently unexploited antiepileptic drug targets.
Assuntos
Epilepsias Parciais/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hipocampo/metabolismo , Epilepsias Parciais/etiologia , Epilepsias Parciais/metabolismo , Epilepsias Parciais/patologia , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Hipocampo/patologia , Humanos , Transdução de SinaisRESUMO
Dysregulated metabolism is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here we report the development of a chemical approach to detect the oncometabolite fumarate. Our strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddition of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. We demonstrate hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, we show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. Our studies define a novel intersection of bioorthogonal chemistry and metabolite reactivity that may be harnessed to enable biological profiling, imaging, and diagnostic applications.
Assuntos
Alcenos/metabolismo , Carcinoma de Células Renais/metabolismo , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Iminas/metabolismo , Neoplasias Renais/metabolismo , Alcenos/química , Carcinoma de Células Renais/patologia , Fumaratos/análise , Humanos , Iminas/química , Neoplasias Renais/patologia , Estrutura MolecularRESUMO
Iron-sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.
Assuntos
Acidose Láctica/congênito , Fatores de Crescimento de Fibroblastos/metabolismo , Hidroximetilglutaril-CoA Sintase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/congênito , Fatores de Transcrição/genética , Acidose Láctica/genética , Acidose Láctica/metabolismo , Acidose Láctica/patologia , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Metabolismo Energético , Feminino , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Proteínas Ferro-Enxofre/genética , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Understanding the factors that drive recurrence and radiosensitivity in brain metastases would improve prediction of outcomes, treatment planning and development of therapeutics. We investigated the expression of known metastasis-inducing proteins in human brain metastases. METHODS: Immunohistochemistry on metastases removed at neurosurgery from 138 patients to determine the degree and pattern of expression of the proteins S100A4, S100P, AGR2, osteopontin (OPN) and the DNA repair marker FANCD2. Validation of significant findings in a separate prospective series with the investigation of intra-tumoral heterogeneity using image-guided sampling. Assessment of S100A4 expression in brain metastatic and non-metastatic primary breast carcinomas. RESULTS: There was widespread staining for OPN, S100A4, S100P and AGR2 in human brain metastases. Positive staining for S100A4 was independently associated with a shorter time to intracranial progression after resection in multivariate analysis (hazard ratio for negative over positive staining=0.17, 95% CI: 0.04-0.74, P=0.018). S100A4 was expressed at the leading edge of brain metastases in image guided sampling and overexpressed in brain metastatic vs non-brain metastatic primary breast carcinomas. Staining for OPN was associated with a significant increase in survival time after post-operative whole-brain radiotherapy in retrospective (OPN negative 3.43 months, 95% CI: 1.36-5.51 vs OPN positive, 11.20 months 95% CI: 7.68-14.72, Log rank test, P<0.001) and validation populations. CONCLUSIONS: Proteins known to be involved in cellular adhesion and migration in vitro, and metastasis in vivo are significantly expressed in human brain metastases and may be useful biomarkers of intracranial progression and radiosensitivity.
Assuntos
Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundário , Proteínas de Ligação ao Cálcio/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Neoplasias/metabolismo , Osteopontina/metabolismo , Proteínas/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Biópsia Guiada por Imagem , Masculino , Pessoa de Meia-Idade , Mucoproteínas , Proteínas Oncogênicas , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Análise de Sobrevida , Adulto JovemRESUMO
OBJECTIVE: Many different gene families are currently being investigated for their potential role in epilepsy and in the response to antiepileptic drugs. A common research challenge is identifying the members of a gene family that are most significantly dysregulated within the human epileptic focus, before taking them forward for resource-intensive functional studies. Published data about transcriptomic changes within the human epileptic focus remains incomplete. A need exists for an accurate in silico system for the prediction of dysregulated genes within the epileptic focus. We present such a bioinformatic system. We demonstrate the validity of our approach by applying it to the solute carrier (SLC) gene family. There are >400 known SLCs. SLCs have never been systematically studied in epilepsy. METHODS: Using our in silico system, we predicted the SLCs likely to be dysregulated in the epileptic focus. We validated our in silico predictions by identifying ex vivo the SLCs dysregulated in epileptic foci, and determining the overlap between our in silico and ex vivo results. For the ex vivo analysis, we used a custom oligonucleotide microarray containing exon probes for all known SLCs to analyze 24 hippocampal samples obtained from surgery for pharmacoresistant mesial temporal lobe epilepsy and 24 hippocampal samples from normal postmortem controls. RESULTS: There was a highly significant (p < 9.99 × 10(-7) ) overlap between the genes identified by our in silico and ex vivo strategies. The SLCs identified were either metal ion exchangers or neurotransmitter transporters, which are likely to play a part in epilepsy by influencing neuronal excitability. SIGNIFICANCE: The identified SLCs are most likely to mediate pharmacoresistance in epilepsy by enhancing the intrinsic severity of epilepsy, but further functional work will be needed to fully evaluate their role. Our successful in silico strategy can be adapted in order to prioritize genes relevant to epilepsy from other gene families.
Assuntos
Proteínas de Transporte de Cátions/genética , Epilepsia/genética , Perfilação da Expressão Gênica , Predisposição Genética para Doença/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Biologia Computacional , Feminino , Testes Genéticos , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Adulto JovemRESUMO
BACKGROUND: Increasingly, biomarkers have been identified that correlate with improved overall and progression-free survival (OS and PFS) in glioblastoma, including MGMT methylation status and mutations in the IDH1 gene. In this study, we investigated the clinical and biological factors associated with long-term survival in glioblastoma patients treated with chemoradiotherapy. METHOD: Demographic and clinical data were collected for all patients with glioblastoma diagnosed between May 2004 and September 2007, treated with chemoradiotherapy and with associated tissue samples available for biomarker analysis. MGMT methylation was determined by pyrosequencing. IDH1 mutation was identified by R132H immunohistochemistry. Univariate Cox regression analysis of factors associated with survival and Kaplan-Meier survival analysis was performed using the SPSS statistics package. RESULTS: One hundred patients were included in the study. Median follow-up was 12.2 months (range 1.6-102.4). Median OS was 12.1 months (95 % CI: 10.8-13.3) and median PFS was 8.2 months (95 % CI: 6.8-9.5). The 2-, 3- and 5-year survival was 18, 9 and 6 % respectively. Three patients are still alive at 7.4, 8.3 and 8.5 years after diagnosis. Cox proportional-hazards regression identified independent prognostic factors for OS, female (p = 0.019), MGMT methylation (p < 0.0001) and IDH1 mutation (p = 0.023), and for PFS, MGMT methylation (p = 0.001) and IDH1 mutation (p = 0.018). Kaplan-Meier survival analysis showed that MGMT(methylated)/IDH1(+ve) was associated with a significantly longer OS 66.8 months (95 % CI: 0.0-167.8) and PFS 16.9 months (95 % CI: 11.1-22.7) when compared with MGMT(methylated)/IDH1(-ve) OS 15.5 months (95 % CI: 11.6-19.4) and PFS 9.4 months (95 % CI: 8-10.8) (log-rank, P = 0.000) and MGMT(unmethylated)/IDH1(-ve) OS 11.1 months (95 % CI: 8.5-13.7) and PFS 6.3 months (95 % CI: 4.4-8.3) (log-rank, p = 0.000). CONCLUSIONS: While the importance of MGMT methylation is well established, we demonstrate that the combination of MGMT(methylated)/IDH1(+ve) is associated with considerably longer OS and PFS in this series of chemoradiotherapy-treated glioblastoma tumours. The long-term cognitive function and quality of life in these long-term survivors warrant investigation.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Isocitrato Desidrogenase/genética , Mutação , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Intervalo Livre de Doença , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras GenéticasRESUMO
Hypoxic tumor microenvironments pose a significant challenge in cancer treatment. Hypoxia-activated prodrugs like evofosfamide aim to specifically target and eliminate these resistant cells. However, their effectiveness is often limited by reoxygenation after cell death. We hypothesized that ascorbate's pro-oxidant properties could be harnessed to induce transient hypoxia, enhancing the efficacy of evofosfamide by overcoming reoxygenation. To test this hypothesis, we investigated the sensitivity of MIA Paca-2 and A549 cancer cells to ascorbate in vitro and in vivo. Ascorbate induced a cytotoxic effect at 5 mM that could be alleviated by endogenous administration of catalase, suggesting a role for hydrogen peroxide in its cytotoxic mechanism. In vitro, Seahorse experiments indicated that the generation of hydrogen peroxide consumes oxygen, which is offset at later time points by a reduction in oxygen consumption due to hydrogen peroxide's cytotoxic effect. In vivo, photoacoustic imaging showed pharmacologic ascorbate treatment at sublethal levels triggered a complex, multi-phasic response in tumor oxygenation across both cell lines. Initially, ascorbate generated transient hypoxia within minutes through hydrogen peroxide production, via reactions that consume oxygen. This initial hypoxic phase peaked at around 150 s and then gradually subsided. However, at longer time scales (approximately 300 s) a vasodilation effect triggered by ascorbate resulted in increased blood flow and subsequent reoxygenation. Combining sublethal levels of i. p. Ascorbate with evofosfamide significantly prolonged tumor doubling time in MIA Paca-2 and A549 xenografts compared to either treatment alone. This improvement, however, was only observed in a subpopulation of tumors, highlighting the complexity of the oxygenation response.
RESUMO
Comprehensive molecular characterization and effective therapy in a rare case of metastatic renal oncocytoma.
Assuntos
Adenoma Oxífilo , Neoplasias Renais , Humanos , Pessoa de Meia-Idade , Adenoma Oxífilo/secundário , Adenoma Oxífilo/patologia , Adenoma Oxífilo/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/tratamento farmacológico , Metástase NeoplásicaRESUMO
Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of an aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxoglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1α, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively affects the levels of N6-methyladenosine (m6A), while the levels of 5-formylcytosine (f5C) in mitochondrial tRNA are unaffected. This supports the hypothesis of a differential impact of fumarate accumulation on distinct RNA demethylases. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.
RESUMO
BACKGROUND: ISCU myopathy is a disease caused by muscle-specific deficiency of the Fe-S cluster scaffold protein ISCU. RESULTS: MyoD expression enhanced ISCU mRNA mis-splicing, and oxidative stress exacerbated ISCU depletion in patient cells. CONCLUSION: ISCU protein deficiency in patients results from muscle-specific mis-splicing as well as oxidative stress. SIGNIFICANCE: Oxidative stress negatively influences the mammalian Fe-S cluster assembly machinery by destabilization of ISCU. Iron-sulfur (Fe-S) cluster cofactors are formed on the scaffold protein ISCU. ISCU myopathy is a disease caused by an intronic mutation that leads to abnormally spliced ISCU mRNA. We found that two predominant mis-spliced ISCU mRNAs produce a truncated and short-lived ISCU protein product in multiple patient cell types. Expression of the muscle-specific transcription factor MyoD further diminished normal splicing of ISCU mRNA in patient myoblasts, demonstrating that the process of muscle differentiation enhances the loss of normal ISCU mRNA splicing. ISCU protein was nearly undetectable in patient skeletal muscle, but was higher in patient myoblasts, fibroblasts, and lymphoblasts. We next treated patient cells with pro-oxidants to mimic the oxidative stress associated with muscle activity. Brief hydrogen peroxide treatment or incubation in an enriched oxygen atmosphere led to a marked further reduction of ISCU protein levels, which could be prevented by pretreatment with the antioxidant ascorbate. Thus, we conclude that skeletal muscle differentiation of patient cells causes a higher degree of abnormal ISCU splicing and that oxidative stress resulting from skeletal muscle work destabilizes the small amounts of normal ISCU protein generated in patient skeletal muscles.
Assuntos
Diferenciação Celular , Proteínas Ferro-Enxofre/genética , Doenças Mitocondriais/metabolismo , Músculo Esquelético/citologia , Estresse Oxidativo , Splicing de RNA , Adulto , Idoso , Animais , Feminino , Humanos , Proteínas Ferro-Enxofre/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Especificidade de Órgãos , Adulto JovemRESUMO
Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.
Assuntos
Ferredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Família Multigênica , Oxirredutases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Citosol/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Heme/deficiência , Humanos , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Oxirredutases/genética , Interferência de RNA , Alinhamento de SequênciaRESUMO
OBJECTIVE: To characterize the clinical manifestations and genetic basis of a familial cancer syndrome in patients with lipomas and Birt-Hogg-Dubé-like clinical manifestations including fibrofolliculomas and trichodiscomas and kidney cancer. METHODS: Genomic analysis of blood and renal tumor DNA was performed. Inheritance pattern, phenotypic manifestations, and clinical and surgical management were documented. Cutaneous, subcutaneous, and renal tumor pathologic features were characterized. RESULTS: Affected individuals were found to be at risk for a highly penetrant and lethal form of bilateral, multifocal papillary renal cell carcinoma. Whole genome sequencing identified a germline pathogenic variant in PRDM10 (c.2029 T>C, p.Cys677Arg), which cosegregated with disease. PRDM10 loss of heterozygosity was identified in kidney tumors. PRDM10 was predicted to abrogate expression of FLCN, a transcriptional target of PRDM10, which was confirmed by tumor expression of GPNMB, a TFE3/TFEB target and downstream biomarker of FLCN loss. In addition, a sporadic papillary RCC from the TCGA cohort was identified with a somatic PRDM10 mutation. CONCLUSION: We identified a germline PRDM10 pathogenic variant in association with a highly penetrant, aggressive form of familial papillary RCC, lipomas, and fibrofolliculomas/trichodiscomas. PRDM10 loss of heterozygosity and elevated GPNMB expression in renal tumors indicate that PRDM10 alteration leads to reduced FLCN expression, driving TFE3-induced tumor formation. These findings suggest that individuals with Birt-Hogg-Dubé-like manifestations and subcutaneous lipomas, but without a germline pathogenic FLCN variant, should be screened for germline PRDM10 variants. Importantly, kidney tumors identified in patients with a pathogenic PRDM10 variant should be managed with surgical resection instead of active surveillance.