RESUMO
Despite the higher incidence of cancer with increasing age, few preclinical or clinical studies incorporate age. This, coupled with an aging world population, requires that we improve our understanding of how aging affects cancer development, progression, and treatment. One key area will be how the tumor microenvironment (TME) changes with age. Metabolite levels are an essential component of the TME, and they are affected by the metabolic requirements of the cells present and systemic metabolite availability. These factors are affected by aging, causing different TME metabolic states between young and older adults. In this review, we will summarize what is known about how aging impacts the TME metabolic state, and suggest how we can improve our understanding of it.
Assuntos
Neoplasias , Microambiente Tumoral , Humanos , Idoso , Neoplasias/terapiaRESUMO
The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Animais , Camundongos , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Asparagina/metabolismo , Adenocarcinoma/tratamento farmacológico , Simbiose , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
EBV gene expression is repressed during viral latency to prevent an immune response, but it is not known how metabolism contributes to this silencing. In this issue of Cell Metabolism, Guo et al. describe how methionine restriction reactivates the expression of EBV genes, offering new therapeutic approaches against EBV-driven diseases.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Metionina , Latência ViralRESUMO
Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Asparagina/metabolismo , Mitocôndrias/metabolismo , Animais , Asparagina/farmacologia , Ácido Aspártico/deficiência , Ácido Aspártico/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dieta/veterinária , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Neoplasias/patologia , Nucleotídeos/metabolismo , Taxa de SobrevidaRESUMO
BACKGROUND: Statins inhibit HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Epidemiological and pre-clinical evidence support an association between statin use and delayed prostate cancer (PCa) progression. Here, we evaluated the effects of neoadjuvant fluvastatin treatment on markers of cell proliferation and apoptosis in men with localized PCa. METHODS: Thirty-three men were treated daily with 80 mg fluvastatin for 4-12 weeks in a single-arm window-of-opportunity study between diagnosis of localized PCa and radical prostatectomy (RP) (ClinicalTrials.gov: NCT01992042). Percent Ki67 and cleaved Caspase-3 (CC3)-positive cells in tumor tissues were evaluated in 23 patients by immunohistochemistry before and after treatment. Serum and intraprostatic fluvastatin concentrations were quantified by liquid chromatography-mass spectrometry. RESULTS: Baseline characteristics included a median prostate-specific antigen (PSA) level of 6.48 ng/mL (IQR: 4.21-10.33). The median duration of fluvastatin treatment was 49 days (range: 27-102). Median serum low-density lipoprotein levels decreased by 35% after treatment, indicating patient compliance. Median PSA decreased by 12%, but this was not statistically significant in our small cohort. The mean fluvastatin concentration measured in the serum was 0.2 µM (range: 0.0-1.1 µM), and in prostatic tissue was 8.5 nM (range: 0.0-77.0 nM). At these concentrations, fluvastatin induced PCa cell death in vitro in a dose- and time-dependent manner. In patients, fluvastatin treatment did not significantly alter intratumoral Ki67 positivity; however, a median 2.7-fold increase in CC3 positivity (95% CI: 1.9-5.0, p = 0.007) was observed in post-fluvastatin RP tissues compared with matched pre-treatment biopsy controls. In a subset analysis, this increase in CC3 was more pronounced in men on fluvastatin for >50 days. CONCLUSIONS: Fluvastatin prior to RP achieves measurable drug concentrations in prostatic tissue and is associated with promising effects on tumor cell apoptosis. These data warrant further investigation into the anti-neoplastic effects of statins in prostate tissue.
Assuntos
Fluvastatina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Idoso , Apoptose , Biomarcadores Tumorais/metabolismo , Caspase 3/metabolismo , Progressão da Doença , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Projetos Piloto , Cuidados Pré-Operatórios , Prostatectomia/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgiaRESUMO
OBJECTIVE: The statin family of cholesterol-lowering drugs has been shown to induce tumor-specific apoptosis by inhibiting the rate-limiting enzyme of the mevalonate (MVA) pathway, HMG-CoA reductase (HMGCR). Accumulating evidence suggests that statin use may delay prostate cancer (PCa) progression in a subset of patients; however, the determinants of statin drug sensitivity in PCa remain unclear. Our goal was to identify molecular features of statin-sensitive PCa and opportunities to potentiate statin-induced PCa cell death. METHODS: Deregulation of HMGCR expression in PCa was evaluated by immunohistochemistry. The response of PCa cell lines to fluvastatin-mediated HMGCR inhibition was assessed using cell viability and apoptosis assays. Activation of the sterol-regulated feedback loop of the MVA pathway, which was hypothesized to modulate statin sensitivity in PCa, was also evaluated. Inhibition of this statin-induced feedback loop was performed using RNA interference or small molecule inhibitors. The achievable levels of fluvastatin in mouse prostate tissue were measured using liquid chromatography-mass spectrometry. RESULTS: High HMGCR expression in PCa was associated with poor prognosis; however, not all PCa cell lines underwent apoptosis in response to treatment with physiologically-achievable concentrations of fluvastatin. Rather, most cell lines initiated a feedback response mediated by sterol regulatory element-binding protein 2 (SREBP2), which led to the further upregulation of HMGCR and other lipid metabolism genes. Overcoming this feedback mechanism by knocking down or inhibiting SREBP2 potentiated fluvastatin-induced PCa cell death. Notably, we demonstrated that this feedback loop is pharmacologically-actionable, as the drug dipyridamole can be used to block fluvastatin-induced SREBP activation and augment apoptosis in statin-insensitive PCa cells. CONCLUSION: Our study implicates statin-induced SREBP2 activation as a PCa vulnerability that can be exploited for therapeutic purposes using clinically-approved agents.
Assuntos
Antineoplásicos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/metabolismo , Neoplasias da Próstata/metabolismo , Esteróis/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dipiridamol/farmacologia , Reposicionamento de Medicamentos , Fluvastatina/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.
Assuntos
Carcinogênese/genética , Expressão Gênica/genética , Histona Metiltransferases/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Epigênese Genética/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genéticaRESUMO
The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.
Assuntos
Proteínas de Transporte/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Metabolismo dos Carboidratos/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Glicólise/fisiologia , Humanos , Ácido Hialurônico/fisiologia , Hialuronoglucosaminidase/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais , Tristetraprolina/metabolismo , Tristetraprolina/fisiologiaRESUMO
The statin family of drugs preferentially triggers tumor cell apoptosis by depleting mevalonate pathway metabolites farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are used for protein prenylation, including the oncoproteins of the RAS superfamily. However, accumulating data indicate that activation of the RAS superfamily are poor biomarkers of statin sensitivity, and the mechanism of statin-induced tumor-specific apoptosis remains unclear. Here we demonstrate that cancer cell death triggered by statins can be uncoupled from prenylation of the RAS superfamily of oncoproteins. Ectopic expression of different members of the RAS superfamily did not uniformly sensitize cells to fluvastatin, indicating that increased cellular demand for protein prenylation cannot explain increased statin sensitivity. Although ectopic expression of HRAS increased statin sensitivity, expression of myristoylated HRAS did not rescue this effect. HRAS-induced epithelial-to-mesenchymal transition (EMT) through activation of zinc finger E-box binding homeobox 1 (ZEB1) sensitized tumor cells to the antiproliferative activity of statins, and induction of EMT by ZEB1 was sufficient to phenocopy the increase in fluvastatin sensitivity; knocking out ZEB1 reversed this effect. Publicly available gene expression and statin sensitivity data indicated that enrichment of EMT features was associated with increased sensitivity to statins in a large panel of cancer cell lines across multiple cancer types. These results indicate that the anticancer effect of statins is independent from prenylation of RAS family proteins and is associated with a cancer cell EMT phenotype.Significance: The use of statins to target cancer cell EMT may be useful as a therapy to block cancer progression. Cancer Res; 78(5); 1347-57. ©2017 AACR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fluvastatina/farmacologia , Neoplasias/patologia , Prenilação de Proteína/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Proteínas ras/metabolismo , Apoptose , Biomarcadores Tumorais , Proliferação de Células , Humanos , Ácido Mevalônico/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Proteínas ras/genéticaRESUMO
The mevalonate (MVA) pathway is an essential metabolic pathway that uses acetyl-CoA to produce sterols and isoprenoids that are integral to tumour growth and progression. In recent years, many oncogenic signalling pathways have been shown to increase the activity and/or the expression of MVA pathway enzymes. This Review summarizes recent advances and discusses unique opportunities for immediately targeting this metabolic vulnerability in cancer with agents that have been approved for other therapeutic uses, such as the statin family of drugs, to improve outcomes for cancer patients.
Assuntos
Redes e Vias Metabólicas , Ácido Mevalônico/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , HumanosRESUMO
Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 µmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Ligases SKP Culina F-Box/metabolismo , Sinvastatina/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade , Linhagem Celular , Relação Dose-Resposta a Droga , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Ratos , Fatores de Tempo , Regulação para CimaRESUMO
The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Ácido Mevalônico/metabolismo , Neoplasias/patologia , Interferência de RNA , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Dimetilaliltranstransferase/genética , Farnesiltranstransferase/genética , Ácidos Graxos Monoinsaturados/química , Feminino , Fluvastatina , Geraniltranstransferase/genética , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Indóis/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a Elemento Regulador de Esterol 2/genéticaRESUMO
Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Estresse Oxidativo/fisiologia , Consumo de Oxigênio , Morte Celular , Respiração Celular , Transporte de Elétrons , Humanos , Tamanho Mitocondrial , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
New therapies are urgently needed for hematologic malignancies, especially in patients with relapsed acute myelogenous leukemia (AML) and multiple myeloma. We and others have previously shown that FDA-approved statins, which are used to control hypercholesterolemia and target the mevalonate pathway (MVA), can trigger tumor-selective apoptosis. Our goal was to identify other FDA-approved drugs that synergize with statins to further enhance the anticancer activity of statins in vivo. Using a screen composed of other FDA approved drugs, we identified dipyridamole, used for the prevention of cerebral ischemia, as a potentiator of statin anticancer activity. The statin-dipyridamole combination was synergistic and induced apoptosis in multiple myeloma and AML cell lines and primary patient samples, whereas normal peripheral blood mononuclear cells were not affected. This novel combination also decreased tumor growth in vivo. Statins block HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the MVA pathway. Dipyridamole blunted the feedback response, which upregulates HMGCR and HMG-CoA synthase 1 (HMGCS1) following statin treatment. We further show that dipyridamole inhibited the cleavage of the transcription factor required for this feedback regulation, sterol regulatory element-binding transcription factor 2 (SREBF2, SREBP2). Simultaneously targeting the MVA pathway and its restorative feedback loop is preclinically effective against hematologic malignancies. This work provides strong evidence for the immediate evaluation of this novel combination of FDA-approved drugs in clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácido Mevalônico/metabolismo , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dipiridamol , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hidroximetilglutaril-CoA Sintase/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size.
Assuntos
Tamanho Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/genética , Animais , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Ensaios de Triagem em Larga Escala , Humanos , Células Jurkat , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatin triggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERα)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genes encoding regulators of mevalonate production, non-sterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Ácidos Graxos Monoinsaturados/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Indóis/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/biossíntese , Feminino , Fluvastatina , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/biossíntese , Células MCF-7 , Ácido Mevalônico/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/biossíntese , Receptor ErbB-2RESUMO
Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack.
Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Humanas/patologia , Mutação/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Western Blotting , Adesão Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio , Fosforilação , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS: The aim of this study was to identify the mechanisms of hypocarnitinemia in patients treated with valproate. METHODS: Plasma concentrations and urinary excretion of carnitine, acetylcarnitine, propionylcarnitine, valproylcarnitine, and butyrobetaine were determined in a patient starting valproate treatment and in 10 patients on long-term valproate treatment. Transport of carnitine and valproylcarnitine by the proximal tubular carnitine transporter OCTN2 was assessed in vitro. RESULTS: In the patient starting valproate, the plasma carnitine and acetylcarnitine levels dropped for 1-3 weeks and had recovered after 3-5 weeks, whereas the plasma levels of propionyl and valproylcarnitine increased steadily over 5 weeks. The renal excretion and excretion fractions (EFs) of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine decreased substantially after starting valproate. Compared with controls, patients on long-term valproate treatment had similar plasma levels of carnitine, acetylcarnitine, and propionylcarnitine, whereas valproylcarnitine was found only in patients. Urinary excretion and renal clearance of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine were decreased in valproate-treated compared with that in control patients, reaching statistical significance for carnitine. The EFs of carnitine, acetylcarnitine, and propionylcarnitine were <5% of the filtered load in controls and were lower in valproate-treated patients. In contrast, the EF for valproylcarnitine approached 100%, resulting from a low affinity of valproylcarnitine for the carnitine transporter OCTN2 and competition with concomitantly filtered carnitine. CONCLUSIONS: The initial drop in plasma carnitine levels of valproate-treated patients is most likely due to impaired carnitine biosynthesis, whereas the recovery of the plasma carnitine levels is explainable by an increased renal expression of OCTN2. Renally excreted valproylcarnitine does not affect renal handling of carnitine in vivo.
Assuntos
Carnitina/sangue , Carnitina/urina , Ácido Valproico/administração & dosagem , Acetilcarnitina/sangue , Acetilcarnitina/urina , Adulto , Betaína/análogos & derivados , Betaína/sangue , Transporte Biológico/efeitos dos fármacos , Carnitina/análogos & derivados , Linhagem Celular , Esquema de Medicação , Feminino , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10µM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.