RESUMO
Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh â E2F1 â PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.
Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Células-Tronco Neurais/metabolismo , PPAR gama/metabolismo , Anilidas/farmacologia , Animais , Animais Recém-Nascidos , Compostos Azo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Células Cultivadas , Neoplasias Cerebelares/diagnóstico por imagem , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Hexoquinase/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Meduloblastoma/diagnóstico por imagem , Meduloblastoma/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuropeptídeos/genética , Tomografia por Emissão de Pósitrons , Hormônios Tireóideos/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transdução Genética/métodos , Proteínas de Ligação a Hormônio da TireoideRESUMO
Development of the cerebellum occurs postnatally and is marked by a rapid proliferation of cerebellar granule neuron precursors (CGNPs). CGNPs are the cells of origin for SHH-driven medulloblastoma, the most common malignant brain tumor in children. Here, we investigated the role of ERK, JNK, and p38 mitogen-activated protein kinases in CGNP proliferation. We found high levels of p38α in proliferating CGNPs. Concomitantly, members of the p38 pathway, such as ASK1, MKK3 and ATF-2, were also elevated. Inhibition of the Shh pathway or CGNP proliferation blunts p38α levels, irrespective of Shh treatment. Strikingly, p38α levels were high in vivo in the external granule layer of the postnatal cerebellum, Shh-dependent mouse medulloblastomas and human medulloblastomas of the SHH subtype. Finally, knocking down p38α by short hairpin RNA-carrying lentiviruses as well as the pharmacologically inhibiting of its kinase activity caused a marked decrease in CGNP proliferation, underscoring its requirement for Shh-dependent proliferation in CGNPs. The inhibition of p38α also caused a decrease in Gli1 and N-myc transcript levels, consistent with reduced proliferation. These findings suggest p38 inhibition as a potential way to increase the efficacy of treatments available for malignancies associated with deregulated SHH signaling, such as basal cell carcinoma and medulloblastoma.
Assuntos
Proliferação de Células/fisiologia , Cerebelo/enzimologia , Células-Tronco Neurais/enzimologia , Neurônios/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Encéfalo/enzimologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/enzimologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Development of the cerebellum, a brain region regulating posture and coordination, occurs post-natally and is marked by rapid proliferation of granule neuron precursors (CGNPs), stimulated by mitogenic Sonic hedgehog (Shh) signaling. ß-Arrestin (ßArr) proteins play important roles downstream of Smoothened, the Shh signal transducer. However, whether Shh regulates ßArrs and what role they play in Shh-driven CGNP proliferation remains to be determined. Here, we report that Shh induces ßArr1 accumulation and localization to the nucleus, where it participates in enhancing expression of the cyclin dependent kinase (cdk) inhibitor p27, whose accumulation eventually drives CGNP cell cycle exit. ßArr1 knockdown enhances CGNP proliferation and reduces p27 expression. Thus, Shh-mediated ßArr1 induction represents a novel negative feedback loop within the Shh mitogenic pathway, such that ongoing Shh signaling, while required for CGNPs to proliferate, also sets up a cell-intrinsic clock programming their ultimate exit from the cell cycle.
Assuntos
Arrestinas/metabolismo , Ciclo Celular/fisiologia , Proteínas Hedgehog/metabolismo , Mitose/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Transdução de Sinais/fisiologia , Animais , Arrestinas/genética , Células Cultivadas , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Hedgehog/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Regiões Promotoras Genéticas , beta-Arrestina 1 , beta-Arrestinas , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Certain types of medulloblastoma, the most common solid pediatric cancer, are proposed to arise from neural precursors known as cerebellar granule neuron precursors (CGNPs), which require signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) for their proliferation and survival. Aberrant activity of these pathways is implicated in medulloblastoma. IGF activates the mammalian Target of Rapamycin (mTOR), a growth-promoting kinase normally kept in check by the tumor suppressive Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2. TSC also counteracts proliferation by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1), preventing progression through G(1)- to S-phase of the cell cycle. We reported that mice with impaired TSC activity show increased susceptibility to Shh-mediated medulloblastoma. CGNPs and tumors from these mice display increased proliferation, mTOR pathway activation, glycogen synthase kinase-3 (GSK-3) alpha/beta inactivation, and atypical p27(Kip1) cytoplasmic localization. GSK-3alpha/beta inactivation was mTOR-dependent, whereas p27(Kip1) localization was uncoupled from mTOR, and was instead regulated by TSC2. These results provide insight into the molecular 'hardwiring' of the mitogenic network downstream of Shh signaling and emphasize the separate yet synergistic effects regulated by the TSC complex in (1) fueling proliferation through mTOR activation/GSK-3alpha/beta inactivation and (2) compromising checkpoint mechanisms via TSC2-dependent p27(Kip1) nuclear exclusion. Future medulloblastoma therapies targeting Shh signaling can be developed to selectively modulate these activities, to restore checkpoint control and attenuate uncontrolled hyperproliferation.
Assuntos
Inativação Gênica , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos , Modelos Biológicos , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Loss of the transcription factor E2F1 elicits a complex metabolic phenotype in mice underscored by reduced adiposity and protection from high fat diet-induced diabetes. Here, we demonstrate that E2F1 directly regulates the gene encoding PDK4 (pyruvate dehydrogenase kinase 4), a key nutrient sensor and modulator of glucose homeostasis that is chronically elevated in obesity and diabetes and acutely induced under the metabolic stress of starvation or fasting. We show that loss of E2F1 in vivo blunts PDK4 expression and improves myocardial glucose oxidation. The absence of E2F1 also corresponds to lower blood glucose levels, improved plasma lipid profile, and increased sensitivity to insulin stimulation. Consistently, enforced E2F1 expression up-regulates PDK4 levels and suppresses glucose oxidation in C(2)C(12) myoblasts. Furthermore, inactivation of Rb, the repressor of E2F-dependent transcription, markedly induces PDK4 and triggers the enrichment of E2F1 occupancy onto the PDK4 promoter as detected by chromatin immunoprecipitation analysis. Two overlapping E2F binding sites were identified on this promoter. Transactivation assays later verified E2F1 responsiveness of this promoter element in C(2)C(12) myoblasts and IMR90 fibroblasts, an effect that was completely abrogated following mutation of the E2F sites. Taken together, our data illustrate how the E2F1 mitogen directly regulates PDK4 levels and influences cellular bioenergetics, namely mitochondrial glucose oxidation. These results are relevant to the pathophysiology of chronic diseases like obesity and diabetes, where PDK4 is dysregulated and could have implications pertinent to the etiology of tumor metabolism, especially in cancers with Rb pathway defects.
Assuntos
Fator de Transcrição E2F1/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteína do Retinoblastoma/metabolismo , Adiposidade/fisiologia , Animais , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Gorduras na Dieta/metabolismo , Fator de Transcrição E2F1/genética , Jejum/metabolismo , Feminino , Fibroblastos/enzimologia , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Complexos Multiproteicos/genética , Mutação , Mioblastos/enzimologia , Miocárdio/enzimologia , Neoplasias/enzimologia , Neoplasias/genética , Oxirredução , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteína do Retinoblastoma/genética , Inanição/enzimologia , Inanição/genética , Regulação para Cima/fisiologiaRESUMO
The transcription factor FoxO1 contributes to the metabolic adaptation to fasting by suppressing muscle oxidation of glucose, sparing it for glucose-dependent tissues. Previously, we reported that FoxO1 activation in C(2)C(12) muscle cells recruits the fatty acid translocase CD36 to the plasma membrane and increases fatty acid uptake and oxidation. This, together with FoxO1 induction of lipoprotein lipase, would promote the reliance on fatty acid utilization characteristic of the fasted muscle. Here, we show that CD36-mediated fatty acid uptake, in turn, up-regulates protein levels and activity of FoxO1 as well as its target PDK4, the negative regulator of glucose oxidation. Increased fatty acid flux or enforced CD36 expression in C(2)C(12) cells is sufficient to induce FoxO1 and PDK4, whereas CD36 knockdown has opposite effects. In vivo, CD36 loss blunts fasting induction of FoxO1 and PDK4 and the associated suppression of glucose oxidation. Importantly, CD36-dependent regulation of FoxO1 is mediated by the nuclear receptor PPARdelta/beta. Loss of PPARdelta/beta phenocopies CD36 deficiency in blunting fasting induction of muscle FoxO1 and PDK4 in vivo. Expression of PPARdelta/beta in C(2)C(12) cells, like that of CD36, robustly induces FoxO1 and suppresses glucose oxidation, whereas co-expression of a dominant negative PPARdelta/beta compromises FoxO1 induction. Finally, several PPRE sites were identified in the FoxO1 promoter, which was responsive to PPARdelta/beta. Agonists of PPARdelta/beta were sufficient to confer responsiveness and transactivate the heterologous FoxO1 promoter but not in the presence of dominant negative PPARdelta/beta. Taken together, our findings suggest that CD36-dependent FA activation of PPARdelta/beta results in the transcriptional regulation of FoxO1 as well as PDK4, recently shown to be a direct PPARdelta/beta target. FoxO1 in turn can regulate CD36, lipoprotein lipase, and PDK4, reinforcing the action of PPARdelta/beta to increase muscle reliance on FA. The findings could have implications in the chronic abnormalities of fatty acid metabolism associated with obesity and diabetes.
Assuntos
Adaptação Fisiológica , Antígenos CD36/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Músculos/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Proteínas Quinases/metabolismo , Animais , Sequência de Bases , Antígenos CD36/genética , Linhagem Celular , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Glucose/metabolismo , Cinética , Camundongos , Camundongos Knockout , Oxirredução , Transcrição Gênica/genética , Regulação para CimaRESUMO
Although the human genome has been sequenced, progress in understanding gene regulation in humans has been particularly slow. Many computational approaches developed for lower eukaryotes to identify cis-regulatory elements and their associated target genes often do not generalize to mammals, largely due to the degenerate and interactive nature of such elements. Motivated by the switch-like behavior of transcriptional responses, we present a systematic approach that allows adaptive determination of active transcriptional subnetworks (cis-motif combinations, the direct target genes and physiological processes regulated by the corresponding transcription factors) from microarray data in mammals, with accuracy similar to that achieved in lower eukaryotes. Our analysis uncovered several new subnetworks active in human liver and in cell-cycle regulation, with similar functional characteristics as the known ones. We present biochemical evidence for our predictions, and show that the recently discovered G2/M-specific E2F pathway is wider than previously thought; in particular, E2F directly activates certain mitotic genes involved in hepatocellular carcinomas. Additionally, we demonstrate that this method can predict subnetworks in a condition-specific manner, as well as regulatory crosstalk across multiple tissues. Our approach allows systematic understanding of how phenotypic complexity is regulated at the transcription level in mammals and offers marked advantage in systems where little or no prior knowledge of transcriptional regulation is available.
Assuntos
Regulação da Expressão Gênica , Modelos Genéticos , Fatores de Transcrição/genética , Transcrição Gênica , Algoritmos , Animais , Ciclo Celular/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Genômica , Fator 1 Nuclear de Hepatócito/genética , Fator 1 Nuclear de Hepatócito/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Emerging evidence documents a key function for the forkhead transcription factor FoxO1 in cellular metabolism. Here, we investigate the role of FoxO1 in the regulation of fatty acid (FA) metabolism in muscle cells. C2C12 cells expressing an inducible construct with either wild type FoxO1 or a mutant form (FoxO1/TSS) refractory to the protein kinase B inhibitory effects were generated. FoxO1 activation after myotube formation altered the expression of several genes of FA metabolism. Acyl-CoA oxidase and peroxisome proliferator-activated receptor delta mRNA levels increased 2.2-fold and 1.4-fold, respectively, whereas mRNA for acetyl-CoA carboxylase decreased by 50%. Membrane uptake of oleate increased 3-fold, and oleate oxidation increased 2-fold. Cellular triglyceride content was also increased. The enhanced FA utilization induced by FoxO1 was mediated by a severalfold increase in plasma membrane level of the fatty acid translocase FAT/CD36 and eliminated by cell treatment with the CD36 inhibitor sulfo-N-succinimidyl-oleate. We conclude that FoxO1 activation induces coordinate increases in FA uptake and oxidation and that these effects are mediated, at least in part, by membrane enrichment in CD36. The data suggest that FoxO1 contributes to preparing the muscle cell for the increased reliance on FA metabolism that is characteristic of fasting. Dysregulation of FoxO1 in muscle could contribute to intramuscular lipid accumulation and insulin resistance by maintaining activation of FA uptake.
Assuntos
Antígenos CD36/metabolismo , Ácidos Graxos/metabolismo , Mioblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transporte Biológico/fisiologia , Restrição Calórica , Linhagem Celular , Ácidos Graxos/química , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica , Glucose/metabolismo , Camundongos , Mioblastos/citologia , Oxirredução , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Mitose/fisiologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Aneuploidia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromossomos Humanos , Fatores de Transcrição E2F , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Proteínas Mad2 , Camundongos , Mutação/genética , Proteínas Nucleares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
PURPOSE OF REVIEW: This review highlights recent advances related to malignancies in the field of peroxisome proliferator-activated receptors (PPARs). It also discusses the implications of cancer research and therapy. RECENT FINDINGS: In the last few years, genetic evidence has implicated the PPARs, specifically PPARgamma and PPARbeta/delta, in tumorigenesis. Also, new insights into the regulation of the nuclear hormone receptors have emerged. SUMMARY: Exciting research in PPAR biology has established these nuclear factors as key regulators of metabolism and energy homeostasis. Evidence indicates that PPARs can also affect the pathogenesis and development of tumors. However, the type of effects observed thus far appears to depend on the experimental context. As a result, the findings are generating much debate, as PPAR agonists are widespread targets in the treatment of metabolic disorders such as diabetes and dyslipidemia. Here, we summarize the most recent advances in this field, outline the conflicting reports and discuss their overall implications in cancer research.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Neoplasias/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Animais , Humanos , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , PesquisaRESUMO
Unrestrained E2F activity forces S phase entry and promotes apoptosis through p53-dependent and -independent mechanisms. Here, we show that deregulation of E2F by adenovirus E1A, loss of Rb or enforced E2F-1 expression results in the accumulation of caspase proenzymes through a direct transcriptional mechanism. Increased caspase levels seem to potentiate cell death in the presence of p53-generated signals that trigger caspase activation. Our results demonstrate that mitogenic oncogenes engage a tumour suppressor network that functions at multiple levels to efficiently induce cell death. The data also underscore how cell cycle progression can be coupled to the apoptotic machinery.