RESUMO
Introduction: Reprogramming of cellular metabolism is now a hallmark of tumorigenesis. In recent years, research on pancreatic neuroendocrine tumors (pNETs) has focused on genetic and epigenetic modifications and related signaling pathways, but few studies have been devoted to characterizing the metabolic profile of these tumors. In this review, we thoroughly investigate the metabolic pathways in pNETs by analyzing the transcriptomic and metabolomic data available in the literature. Methodology: We retrieved and downloaded gene expression profiles from all publicly available gene set enrichments (GSE43797, GSE73338, and GSE117851) to compare the differences in expressed genes based on both the stage and MEN1 mutational status. In addition, we conducted a systematic review of metabolomic data in NETs. Results: By combining transcriptomic and metabolomic approaches, we have identified a distinctive metabolism in pNETs compared with controls without pNETs. Our analysis showed dysregulations in the one-carbon, glutathione, and polyamine metabolisms, fatty acid biosynthesis, and branched-chain amino acid catabolism, which supply the tricarboxylic acid cycle. These targets are implicated in pNET cell proliferation and metastasis and could also have a prognostic impact. When analyzing the profiles of patients with or without metastasis, or with or without MEN1 mutation, we observed only a few differences due to the scarcity of published clinical data in the existing research. Consequently, further studies are now necessary to validate our data and investigate these potential targets as biomarkers or therapeutic solutions, with a specific focus on pNETs.
Assuntos
Tumores Neuroectodérmicos Primitivos , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Tumores Neuroendócrinos/patologia , Prognóstico , Epigênese Genética , Tumores Neuroectodérmicos Primitivos/genéticaRESUMO
CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) with SDHx pathogenic variants (PVs) are characterized by a higher intratissular succinate/fumarate ratio (RS/F) than non-SDHx-mutated ones. Also, an increase in serum succinate levels has been reported in patients with germline SDHB or SDHD PV. OBJECTIVE: To assess whether measurement of serum succinate, fumarate levels, and RS/F might aid identification of an SDHx germline PV/likely pathogenic variant (LPV) in patients with PPGL or in asymptomatic relatives; and to guide identification of a PV/LPV among the variants of unknown significance (VUS) identified in SDHx by next-generation sequencing. METHODS: This prospective monocentric study included 93 patients attending an endocrine oncogenetic unit for genetic testing. Succinate and fumarate were measured in serum by gas chromatography coupled to mass spectrometry. The RS/F was calculated to assess SDH enzymatic function. Diagnostic performance was assessed by receiver operating characteristic analysis. RESULTS: RS/F had a higher discriminant power than succinate alone to identify an SDHx PV/LPV in patients with PPGL. However, SDHD PVs/LPVs are frequently missed. Only RS/F differed between asymptomatic SDHB/SDHD PV/LPV carriers and SDHB/SDHD-linked patients with PPGL. Finally RS/F could be helpful to easily evaluate the functional impact of VUS in SDHx. CONCLUSION: Measurement of serum RS/F in patients with PPGL and in asymptomatic relatives is a valuable initial workup tool to detect those carrying a germline PV/LPV in SDHx. Its discriminative power is equal or superior to those of succinate measured alone. SDHD PVs/LPVs are less frequently identified by these biochemical tools. Use of RS/F for SDHx VUS reclassification needs to be evaluated further.
Assuntos
Neoplasias das Glândulas Suprarrenais , Paraganglioma , Feocromocitoma , Humanos , Feocromocitoma/diagnóstico , Feocromocitoma/genética , Feocromocitoma/patologia , Ácido Succínico , Fumaratos , Estudos Prospectivos , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Paraganglioma/diagnóstico , Paraganglioma/genética , Paraganglioma/patologia , Carcinogênese , Transformação Celular Neoplásica , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Mutação em Linhagem GerminativaRESUMO
Apart from the oncometabolite succinate, little studies have appeared on extra-mitochondrial pathways in Succinate Dehydrogenase (SDH) genetic deficiency. The role of NADH/NAD+ redox status and dependent pathways was recently emphasized. Therein, fatty acid (FA) metabolism data were collected here in 30 patients with a loss of function (LOF) variant in one SDHx gene (either with a pheochromocytoma/paraganglioma (PPGL) or asymptomatic) and in 22 wild-type SDHx controls (with PPGL or asymptomatic). Blood acylcarnitines in two patients, peroxisomal biomarkers, very long-chain saturated FA (VLCFA), and C20 to C24 n-3 polyunsaturated fatty acids (PUFA), in all patients were measured by mass spectrometry. Preliminary data showed elevated even and odd long- and very long-chain acylcarnitines in two patients with a SDHB variant. In the whole series, no abnormalities were observed in biomarkers of peroxisomal ß-oxidation (C27-bile acids, VLCFAs and phytanic/pristanic acids) in SDHx patients. However, an increased hexaene to pentaene PUFA ratio ([TetraHexaenoic Acid + DocosaHexaenoic Acid]/[n-3 DocosaPentaenoic Acid + EicosaPentaenoic Acid]) was noticed in patients with SDHC/SDHD variants vs patients with SDHA/SDHB variants or controls, suggesting a higher degree of unsaturation of PUFAs. Within the group with a SDHx variant, Eicosapentaenoate/Tetracosahexaenoate ratio, as an empiric index of shortening/elongation balance, discriminated patients with PPGL from asymptomatic ones. Present findings argue for stimulated elongation of saturated FAs, changes in shortening/elongation balance and desaturation rates of C20-C24 PUFAs in SDH-deficient patients with PPGL. Overall, oxidation of NADH sustained by these pathways might reflect or impact glycolytic NAD+ recycling and hence tumor proliferation.
Assuntos
Neoplasias das Glândulas Suprarrenais , Ácidos Graxos/sangue , Paraganglioma , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/genética , Ácidos e Sais Biliares , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Complexo II de Transporte de Elétrons/deficiência , Humanos , Erros Inatos do Metabolismo , Doenças Mitocondriais , Mutação , NAD/metabolismo , Paraganglioma/genética , Paraganglioma/metabolismo , Paraganglioma/patologia , Feocromocitoma/genética , Succinato Desidrogenase/genética , Ácido Succínico/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a longstanding issue in clinical practice and metabolic research. New clues in better understanding the pathogenesis of HCC might relate to the metabolic context in patients with citrin (aspartate-glutamate carrier 1) deficiency (CD). Because citrin-deficient liver (CDL) is subject to HCC, it represents a unique metabolic model to highlight the mechanisms of HCC promotion, offering different angles of study than the classical metabolic syndrome/obesity/non-alcoholic fatty liver disease (NAFLD)/HCC study axis. In turn, the metabolic features of HCC could shed light on the pathogenesis of CDL. Among these, HCC-induced re-activation of aralar-1 (aspartate-glutamate carrier 2), physiologically not expressed in the adult liver, might take place in CDL, so gene redundancy for mitochondrial aspartate-glutamate carriers would be exploited by the CDL. This proposed (aralar-1 re-activation) and known (citrate/malate cycle) adaptive mechanisms may substitute for the impaired function in CD and are consistent with the clinical remission stage of CD and CD improvement by medium-chain triglycerides (MCT). However, these metabolic adaptive benefits could also promote HCC development. In CD, as a result of PPARα down-regulation, liver mitochondrial fatty acid-derived acetyl-CoA would, like glucose-derived acetyl-CoA, be used for lipid anabolism and fuel nuclear acetylation events which might trigger aralar-1 re-activation as seen in non-CD HCC. A brief account of these metabolic events which might lead to aralar-1 re-activation in CDL is here given. Consistency of this account for CDL events further relies on the protective roles of PPARα and inhibition of mitochondrial and plasma membrane citrate transporters in non-CD HCC.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ânions Orgânicos/deficiência , Acetilcoenzima A/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Humanos , NAD/metabolismo , Triglicerídeos/metabolismoRESUMO
The mechanisms whereby the Hippo pathway effector YAP regulates cancer cell stemness, plasticity, and chemoresistance are not fully understood. We previously showed that in 5-fluorouracil (5-FU)-resistant colorectal cancer cells, the transcriptional coactivator YAP is differentially regulated at critical transitions connected with reversible quiescence/dormancy to promote metastasis. Here, we found that experimental YAP activation in 5-FU-sensitive and 5-FU-resistant HT29 colorectal cancer cells enhanced nuclear YAP localization and the transcript levels of the retinoic acid (RA) receptors RARα/γ and RAR target genes CYP26A1, ALDH1A3, and LGR5 through RA Response Elements (RARE). In these two cell models, constitutive YAP activation reinforced the expression of the stemness biomarkers and regulators ALDH1A3, LGR5, and OCT4. Conversely, YAP silencing, RAR/RXR inhibition by the pan-RAR antagonist BMS493, and vitamin A depletion downregulated stemness traits and self-renewal. Regarding the mechanisms engaged, proximity-dependent labeling, nuclear YAP pulldown coupled with mass spectrometry, and chromatin immunoprecipitation (ChIP)/re-ChIP experiments revealed: (i) the nuclear colocalization/interaction of YAP with RARγ and RXRs; and (ii) combined genomic co-occupancy of YAP, RARα/γ, and RXRα interactomes at proximal RAREs of LGR5 and ALDH1A3 promoters. Moreover, activation of the YAP/RAR-RXR cross-talk in colorectal cancer cells promoted RAR self-activation loops via vitamin A metabolism, RA, and active RAR ligands generated by ALDH1A3. Together, our data identify YAP as a bona fide RAR-RXR transcriptional coactivator that acts through RARE-activated stemness genes. IMPLICATIONS: Targeting the newly identified YAP/RAR-RXR cross-talk implicated in cancer cell stemness maintenance may lead to multitarget combination therapies for patients with colorectal cancer.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Fluoruracila/farmacologia , Células-Tronco Neoplásicas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Células HT29 , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Receptor Cross-Talk , Regulação para CimaRESUMO
The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.
Assuntos
Fibroblastos/patologia , Mitocôndrias/patologia , Doenças Mitocondriais/diagnóstico , Fosforilação Oxidativa , Oxigênio/análise , Adolescente , Biópsia , Técnicas de Cultura de Células , Linhagem Celular , Estudos de Viabilidade , Feminino , Fibroblastos/citologia , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/patologia , Oxigênio/metabolismo , Consumo de Oxigênio , Reprodutibilidade dos Testes , Estudos Retrospectivos , Pele/citologia , Pele/patologiaRESUMO
High resolution oxymetry study (HROS) of skeletal muscle usually requires 90-120 min preparative phase (dissection, permeabilization and washing). This work reports on the suitability of a rapid muscle preparation which by-passes this long preparation. For a few seconds only, muscle biopsy from pigs is submitted to gentle homogenization at 8000 rotations per minute using an ultra-dispersor apparatus. Subsequent HROS is performed using FCCP instead of ADP, compounds crossing and not plasma membrane, respectively. This simplified procedure compares favorably with classical (permeabilized fibers) HROS in terms of respiratory chain complex activities. Mitochondria from cells undergoing ultradispersion were functionally preserved as attested by relative inefficacy of added cytochrome C (not crossing intact mitochondrial outer membrane) to stimulate mitochondrial respiration. Responsiveness of respiration to ADP (in the absence of FCCP) suggested that these intact mitochondria were outside cells disrupted by ultradispersion or within cells permeated by this procedure.
Assuntos
Respiração Celular , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Animais , Biópsia , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Membranas Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Permeabilidade , SuínosRESUMO
In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (p = 0.0041) and decreasing body weight (p < 0.05). A significant increase in regulatory T-cell count (p = 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (p = 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Interleucina-6/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Esclerose Lateral Amiotrófica/genética , Animais , Peso Corporal/genética , Citocinas/sangue , Avaliação da Deficiência , Modelos Animais de Doenças , Seguimentos , Redes Reguladoras de Genes , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Interleucina-6/genética , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.
Assuntos
Amidinotransferases/metabolismo , Creatina/metabolismo , Guanidinoacetato N-Metiltransferase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Amidinotransferases/deficiência , Amidinotransferases/genética , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/deficiência , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animais , Transporte Biológico Ativo , Encefalopatias Metabólicas Congênitas/diagnóstico , Encefalopatias Metabólicas Congênitas/enzimologia , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Creatina/biossíntese , Creatina/deficiência , Creatina/genética , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Metabolismo Energético , Guanidinoacetato N-Metiltransferase/deficiência , Guanidinoacetato N-Metiltransferase/genética , Atrofia Girata/diagnóstico , Atrofia Girata/enzimologia , Atrofia Girata/genética , Atrofia Girata/metabolismo , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/enzimologia , Hiperamonemia/genética , Hiperamonemia/metabolismo , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Transtornos do Desenvolvimento da Linguagem/diagnóstico , Transtornos do Desenvolvimento da Linguagem/enzimologia , Transtornos do Desenvolvimento da Linguagem/genética , Transtornos do Desenvolvimento da Linguagem/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Deficiência Intelectual Ligada ao Cromossomo X/enzimologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Metilação , Proteínas de Transporte da Membrana Mitocondrial , Transtornos dos Movimentos/congênitoRESUMO
PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Fluoruracila/farmacologia , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Fatores de Transcrição/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proliferação de Células , Quinase do Ponto de Checagem 2/metabolismo , Quimioterapia Adjuvante , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/uso terapêutico , Expressão Gênica , Células HT29 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Terapia Neoadjuvante , Micrometástase de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Modelos de Riscos Proporcionais , Transporte Proteico , Proteínas Proto-Oncogênicas c-yes/genética , Fatores de Transcrição/genéticaRESUMO
Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.
Assuntos
Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Transtornos do Metabolismo dos Lipídeos/etiologia , Lipídeos , Fibras Musculares Esqueléticas/patologia , Mioblastos/patologia , Fosfatidato Fosfatase/genética , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Ciclo Celular , Proliferação de Células , Criança , Pré-Escolar , Estresse do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica , Humanos , Transtornos do Metabolismo dos Lipídeos/metabolismo , Transtornos do Metabolismo dos Lipídeos/patologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Associadas a Pancreatite , Fosfatidato Fosfatase/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiólise/etiologia , Rabdomiólise/metabolismo , Rabdomiólise/patologiaRESUMO
This review is aimed at illustrating that mitochondrial dysfunction and altered lipid homeostasis may concur in a variety of pathogenesis states, being either contributive or consecutive to primary disease events. Underlying mechanisms for this concurrence are far from being the exhaustive elements taking place in disease development. They may however complicate, contribute or cause the disease. In the first part of the review, physiological roles of mitochondria in coordinating lipid metabolism and in controlling reactive oxygen species (ROS), ATP and calcium levels are briefly presented. In a second part, clues for how mitochondria-driven alterations in lipid metabolism may induce toxicity are discussed. In the third part, it is illustrated how mitochondrial dysfunction and lipid homeostasis disruption may be associated (i) to complicate type 1 diabetes (pancreatic ß-cell mitochondrial dysfunction in ATP yield induces reduced insulin secretion and hence disruption of glucose and lipid metabolism), (ii) to contribute to type 2 diabetes and other insulin resistant states (mitochondrial impairment may induce adipocyte dysfunction with subsequent increase in circulating free fatty acids and their abnormal deposit in non adipose tissues (pancreatic ß-cells, skeletal muscle and liver) which results in lipotoxicity and mitochondrial dysfunction), (iii) to offer new clues in our understanding of how the brain controls feeding supply and energy expenditure, (iv) to promote cancer development notably via fatty acid oxidation/synthesis imbalance (in favor of synthesis) further strengthened in some cancers by a lipogenetic benefit induced by a HER2/fatty acid synthase cross-talk, and (v) to favor cardiovascular disorders by impacting heart function and arterial wall integrity.
Assuntos
Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Homeostase , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mucina-4/fisiologia , Neoplasias Pancreáticas/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Ligantes , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos SCID , Microscopia Confocal/métodos , Invasividade Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Receptores CXCR4/biossíntese , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Inativação Gênica , Células HT29 , Humanos , Metotrexato/farmacologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fenótipo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
REG4, the latest member of the regenerating gene family, is overexpressed in inflammatory bowel diseases and gastrointestinal carcinomas. To date, its pathophysiologic role has not been well established. Using HT-29 models, we previously identified REG4 as being overexpressed in colorectal tumor cells displaying a drug-resistance phenotype; some also displayed invasive properties. Thus, we investigated the potential functions of REG4 in biological processes involved in colorectal tumor progression such as cell proliferation, migration and invasion. Colon cancer cells secreting REG4 (HT29-5M21, HT29-5F7 and HT29/REG4-8) or not (HT-29, HT29/CT1 and Caco-2/TC7) were used to analyze the autocrine and paracrine effects of REG4. REG4 was continuously secreted into the culture medium of colon cancer cells. REG4 stimulated cell growth in a paracrine manner after 24 h of treatment. Notably, REG4 promoted migration and invasion of tumor cells in both an autocrine and paracrine manner, and these effects were significantly decreased by concomitant treatment with an anti-REG4 antibody. Using pharmacological inhibitors, we showed that PI3K/Akt, PKAs, PKCs and Rho-like GTPases, but not MAPK, are involved in REG4 invasion signals. In addition, REG4 expression was found to be increased in tissues harboring proliferation and migration properties such as the developing intestine and tissues from inflammatory bowel disease, hyperplastic polyps, adenoma and colorectal cancers. In various situations, REG4 expression was not confined to proliferating cells, regenerating cells or cells of the invasive front of metastatic tumors, suggesting that extracellular REG4 may act on epithelial cells in a paracrine manner. Altogether, our results indicate that REG4 is a multifunctional secreted protein which acts on colorectal cancer cells in an autocrine and paracrine manner. According to its biological functions and tissue expression, REG4 may play an important role in the development and progression of colorectal cancer, as well as in intestinal morphogenesis and epithelium restitution.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/biossíntese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno/química , Colo/embriologia , Colo/patologia , Progressão da Doença , Epitélio/embriologia , Epitélio/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mitógenos , Invasividade Neoplásica , Proteínas Associadas a Pancreatite , CicatrizaçãoRESUMO
We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.