RESUMO
Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe class of PBDs-Zellweger spectrum disorder (ZSD)-is caused by mutations in peroxin genes that result in both non-functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter-organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.
Assuntos
Transtornos Peroxissômicos , Síndrome de Zellweger , Humanos , Mitocôndrias/genética , Peroxinas/metabolismo , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/metabolismoRESUMO
BACKGROUND/AIMS: MiRNA-301a-3p is an oncogenic miRNA whose expression is associated with tumor development, metastases and overall poor prognosis. Estrogen receptor α (ERα) is one of the estrogen hormone-activated transcription factors, which regulates a large number of genes and is involved in the mammary gland development. Expression of ERα is considered to be a good indicator for endocrine therapy and breast cancer survival. Loss of ERα in breast cancer patients indicates invasiveness and poor prognosis. In this study, we focus on the regulation of ERα by miR-301a and its role in transition from estrogen-dependent to estrogen-independent breast cancer. METHODS: Expression of miR-301a-3p was measured by qRT-PCR in tumor tissue samples from 111 patients with primary breast carcinoma and in mammospheres representing in vitro model of cancer stem-like cells. Dual reporter luciferase assay and complementary experiments were performed to validate ESR1 as a direct target of miR-301a-3p. The effect of miR-301a-3p on estrogen signaling was evaluated on the level of gene and protein expression and growth response to estrogens. Finally, the effect of miR-301a-3p expression on tumor growth was studied in nude mice. RESULTS: We identified ESR1 as a direct target of miR-301a-3p. Ectopic miR-301a-3p causes a decrease in ESR1 mRNA and protein level and modulates the expression of ERα target genes in ERα positive breast cancer cells. Consistently, miR-301a-3p causes a decrease in sensitivity of MCF7 cells to 17ß-estradiol and inhibits the growth of estrogen dependent tumor in nude mice. Yet, the mice tumors have significantly increased expression of genes related to cancer stem-like cells and epithelial to mesenchymal transition suggesting enrichment of the population of cells with more invasive properties, in line with our observation that miR-301a-3p expression is highly increased in mammospheres which show a decrease in estrogenic signaling. Importantly, miR-301a-3P level is also increased in primary breast cancer samples exhibiting an ER/PR negative phenotype. CONCLUSION: Our results confirm ESR1 as a direct target of miR-301a-3p and suggest that miR-301a-3p likely contributes to development of estrogen independence, which leads to a more invasive phenotype of breast cancer.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de SinaisRESUMO
Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Here we describe the use of mitochondrially targeted deferoxamine (mitoDFO) as a novel approach to preferentially target cancer cells. The agent showed marked cytostatic, cytotoxic, and migrastatic properties in vitro, and it significantly suppressed tumor growth and metastasis in vivo. The underlying molecular mechanisms included (i) impairment of iron-sulfur [Fe-S] cluster/heme biogenesis, leading to destabilization and loss of activity of [Fe-S] cluster/heme containing enzymes, (ii) inhibition of mitochondrial respiration leading to mitochondrial reactive oxygen species production, resulting in dysfunctional mitochondria with markedly reduced supercomplexes, and (iii) fragmentation of the mitochondrial network and induction of mitophagy. Mitochondrial targeting of deferoxamine represents a way to deprive cancer cells of biologically active iron, which is incompatible with their proliferation and invasion, without disrupting systemic iron metabolism. Our findings highlight the importance of mitochondrial iron metabolism for cancer cells and demonstrate repurposing deferoxamine into an effective anticancer drug via mitochondrial targeting. SIGNIFICANCE: These findings show that targeting the iron chelator deferoxamine to mitochondria impairs mitochondrial respiration and biogenesis of [Fe-S] clusters/heme in cancer cells, which suppresses proliferation and migration and induces cell death. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2289/F1.large.jpg.
Assuntos
Carcinogênese/efeitos dos fármacos , Desferroxamina/administração & dosagem , Quelantes de Ferro/administração & dosagem , Ferro/metabolismo , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Carga Tumoral/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Heme/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Neoplasias/patologia , Células PC-3 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the mitochondrial pyruvate carrier (MPC) and the cellular lactate exporter monocarboxylate transporter 4 (MCT4) as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Cardiomegalia/patologia , Insuficiência Cardíaca/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Animais , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte de Ânions/genética , Cardiomegalia/induzido quimicamente , Cardiomegalia/complicações , Insuficiência Cardíaca/etiologia , Coração Auxiliar , Humanos , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ácido Pirúvico/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Esquerda/fisiologiaRESUMO
Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.
In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than 'normal' cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/biossíntese , Mitocôndrias/metabolismo , Mioblastos/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Lipoilação/genética , Camundongos , OxirreduçãoRESUMO
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.
Assuntos
Adenoma/metabolismo , Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ácido Pirúvico/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Drosophila , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Mamárias Animais/tratamento farmacológico , Mesoporfirinas/administração & dosagem , Animais , Etilenoglicóis/administração & dosagem , Etilenoglicóis/química , Feminino , Técnicas de Silenciamento de Genes , Glicóis/química , Humanos , Células MCF-7 , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Mesoporfirinas/química , Camundongos , Paclitaxel/efeitos adversos , Fotoquimioterapia , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/química , Porfirinas/administração & dosagem , Porfirinas/químicaRESUMO
Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 - thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.
Assuntos
Morte Celular/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Tiorredoxinas/genética , Trifosfato de Adenosina/metabolismo , Ciclo Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Respiração Celular , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Glucose/metabolismo , Humanos , Oxirredução , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.
Assuntos
Neoplasias da Mama/genética , Ferro/metabolismo , Células-Tronco Neoplásicas/enzimologia , Neoplasias da Próstata/genética , Transcriptoma , Animais , Antineoplásicos/farmacologia , Transporte Biológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Quelantes de Ferro/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/genética , Células MCF-7 , Masculino , Camundongos Transgênicos , Mitocôndrias/enzimologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fenótipo , Análise de Componente Principal , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Esferoides Celulares , Tamoxifeno/farmacologiaRESUMO
AIMS: To assess the effect of mitochondrially targeted vitamin E (VE) analogs on mitochondrial function and biogenesis. RESULTS: Mitochondrially targeted vitamin E succinate (MitoVES) is an efficient inducer of apoptosis in cancer cells. Here, we show that unlike its untargeted counterpart α-tocopheryl succinate, MitoVES suppresses proliferation of cancer cells at sub-apoptotic doses by way of affecting the mitochondrial DNA (mtDNA) transcripts. We found that MitoVES strongly suppresses the level of the displacement loop transcript followed by those of mtDNA genes coding for subunits of mitochondrial complexes. This process is coupled to the inhibition of mitochondrial respiration, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. In addition, exposure of cancer cells to MitoVES led to decreased expression of TFAM and diminished mitochondrial biogenesis. The inhibition of mitochondrial transcription was replicated in vivo in a mouse model of HER2(high) breast cancer, where MitoVES lowered the level of mtDNA transcripts in cancer cells but not in normal tissue. INNOVATION: Our data show that mitochondrially targeted VE analogs represent a novel class of mitocans that not only induce apoptosis at higher concentrations but also block proliferation and suppress normal mitochondrial function and transcription at low, non-apoptogenic doses. CONCLUSIONS: Our data indicate a novel, selective anti-cancer activity of compounds that act by targeting mitochondria of cancer cells, inducing significant alterations in mitochondrial function associated with transcription of mtDNA-coded genes. These changes subsequently result in the arrest of cell proliferation.
Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/efeitos dos fármacos , Receptor ErbB-2/genética , alfa-Tocoferol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mitocôndrias/fisiologia , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
AIMS: MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia. RESULTS: MiR126 was up-regulated by oxidative stress in nonmalignant mesothelial (Met5A) and MM (H28) cell lines. In Met5A cells, rotenone inhibited MiR126 expression, but mtDNA depletion and hypoxia up-regulated MiR126. However, these various stimuli suppressed the levels of MiR126 in H28 cells. MiR126 affected mitochondrial energy metabolism, reduced mitochondrial respiration, and promoted glycolysis in H28 cells. This metabolic shift, associated with insulin receptor substrate-1 (IRS1)-modulated ATP-citrate lyase deregulation, resulted in higher ATP and citrate production. These changes were linked to the down-regulation of IRS1 by ectopic MiR126, reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promoted the expression of genes involved in gluconeogenesis and oxidative stress defense. These metabolic changes induced hypoxia-inducible factor-1α (HIF1α) stabilization. Consequently, MiR126 suppressed the malignancy of MM cells in vitro, a notion corroborated by the failure of H28(MiR126) cells to form tumors in nude mice. INNOVATION AND CONCLUSION: MiR126 affects mitochondrial energy metabolism, resulting in MM tumor suppression. Since MM is a fatal neoplastic disease with a few therapeutic options, this finding is of potential translational importance.