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1.
Mol Cell ; 82(18): 3333-3349.e9, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35981542

RESUMO

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.


Assuntos
Cromatina , Proteína do Retinoblastoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição AP-1/genética
2.
Nature ; 620(7973): 393-401, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37407818

RESUMO

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.


Assuntos
Citidina Desaminase , Neoplasias Pulmonares , Humanos , Citidina Desaminase/deficiência , Citidina Desaminase/efeitos dos fármacos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Mutação , Resistencia a Medicamentos Antineoplásicos
3.
Cell ; 154(3): 541-55, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23871696

RESUMO

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.


Assuntos
Replicação do DNA , Dosagem de Genes , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/genética , Cromatina/metabolismo , Cromossomos Humanos Par 1 , Instabilidade Genômica , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Fase S
4.
Mol Cell ; 73(5): 985-1000.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30711375

RESUMO

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.


Assuntos
Neoplasias da Mama/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Mutação , Fosforilação Oxidativa , Fosforilação , Ligação Proteica , Proteômica/métodos , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Transcrição Gênica
6.
Bioorg Med Chem ; 106: 117755, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38749343

RESUMO

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.


Assuntos
Nucleotidiltransferases , Bibliotecas de Moléculas Pequenas , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Relação Estrutura-Atividade , Ligação Proteica , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , DNA Polimerase Dirigida por DNA/metabolismo , Camundongos , Síntese de DNA Translesão
7.
Mol Cell ; 64(6): 1015-1017, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27984740

RESUMO

Repetitive DNA elements are essential for genome function; in this issue of Molecular Cell, Ishak et al. (2016) describe a novel mechanism of epigenetic repression at these elements that requires pRB-dependent recruitment of EZH2.


Assuntos
Sequências Repetitivas de Ácido Nucleico , Proteína do Retinoblastoma/genética , DNA , Genoma
8.
Genes Dev ; 30(13): 1492-502, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27401552

RESUMO

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene to be molecularly defined. RB1 mutations occur in almost all familial and sporadic forms of retinoblastoma, and this gene is mutated at variable frequencies in a variety of other human cancers. Because of its early discovery, the recessive nature of RB1 mutations, and its frequency of inactivation, RB1 is often described as a prototype for the class of tumor suppressor genes. Its gene product (pRB) regulates transcription and is a negative regulator of cell proliferation. Although these general features are well established, a precise description of pRB's mechanism of action has remained elusive. Indeed, in many regards, pRB remains an enigma. This review summarizes some recent developments in pRB research and focuses on progress toward answers for the three fundamental questions that sit at the heart of the pRB literature: What does pRB do? How does the inactivation of RB change the cell? How can our knowledge of RB function be exploited to provide better treatment for cancer patients?


Assuntos
Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular/genética , Proliferação de Células/genética , Fatores de Transcrição E2F/metabolismo , Inativação Gênica/fisiologia , Mutação , Neoplasias/genética , Pesquisa/tendências , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética
9.
Nature ; 546(7658): 426-430, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28607489

RESUMO

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.


Assuntos
Ciclina D3/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , Glicólise/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fosfofrutoquinase-1/metabolismo , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Purinas/farmacologia , Purinas/uso terapêutico , Piruvato Quinase/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Genes Dev ; 29(17): 1875-89, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26314710

RESUMO

The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.


Assuntos
Mitocôndrias/metabolismo , Fosforilação Oxidativa , Proteína do Retinoblastoma/genética , Animais , Células Cultivadas , Colo/fisiopatologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pulmão/fisiopatologia , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteômica , Proteína do Retinoblastoma/metabolismo , Estresse Fisiológico/genética , Transcriptoma
11.
Nature ; 539(7629): 390-395, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27799657

RESUMO

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.


Assuntos
Transformação Celular Neoplásica , Metilação de DNA , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Inativação Gênica , Genes Supressores de Tumor , Glicina/metabolismo , Glicólise , Humanos , Camundongos , Ductos Pancreáticos/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Retroelementos/genética , S-Adenosilmetionina/metabolismo , Serina/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Transaminases/metabolismo
12.
Mol Cell ; 53(6): 993-1004, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24613344

RESUMO

Chromosome instability (CIN), a common feature of solid tumors, promotes tumor evolution and increases drug resistance during therapy. We previously demonstrated that loss of the retinoblastoma protein (pRB) tumor suppressor causes changes in centromere structure and generates CIN. However, the mechanism and significance of this change was unclear. Here, we show that defects in cohesion are key to the pRB loss phenotype. pRB loss alters H4K20 methylation, a prerequisite for efficient establishment of cohesion at centromeres. Changes in cohesin regulation are evident during S phase, where they compromise replication and increase DNA damage. Ultimately, such changes compromise mitotic fidelity following pRB loss. Remarkably, increasing cohesion suppressed all of these phenotypes and dramatically reduced CIN in cancer cells lacking functional pRB. These data explain how loss of pRB undermines genomic integrity. Given the frequent functional inactivation of pRB in cancer, conditions that increase cohesion may provide a general strategy to suppress CIN.


Assuntos
Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Proteína do Retinoblastoma/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma Humano , Histonas/metabolismo , Humanos , Metilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Fase S/genética , Transdução de Sinais , Coesinas
13.
J Natl Compr Canc Netw ; : 1-8, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33761455

RESUMO

BACKGROUND: Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) are widely used as first-line therapy for hormone receptor-positive metastatic breast cancer (HR+ MBC). Although abemaciclib monotherapy is also FDA-approved for treatment of disease progression on endocrine therapy, there is limited insight into the clinical activity of abemaciclib after progression on prior CDK4/6i. PATIENTS AND METHODS: We identified patients with HR+ MBC from 6 cancer centers in the United States who received abemaciclib after disease progression on prior CDK4/6i, and abstracted clinical features, outcomes, toxicity, and predictive biomarkers. RESULTS: In the multicenter cohort, abemaciclib was well tolerated after a prior course of CDK4/6i (palbociclib)-based therapy; a minority of patients discontinued abemaciclib because of toxicity without progression (9.2%). After progression on palbociclib, most patients (71.3%) received nonsequential therapy with abemaciclib (with ≥1 intervening non-CDK4/6i regimens), with most receiving abemaciclib with an antiestrogen agent (fulvestrant, 47.1%; aromatase inhibitor, 27.6%), and the remainder receiving abemaciclib monotherapy (19.5%). Median progression-free survival for abemaciclib in this population was 5.3 months and median overall survival was 17.2 months, notably similar to results obtained in the MONARCH-1 study of abemaciclib monotherapy in heavily pretreated HR+/HER2-negative CDK4/6i-naïve patients. A total of 36.8% of patients received abemaciclib for ≥6 months. There was no relationship between the duration of clinical benefit while on palbociclib and the subsequent duration of treatment with abemaciclib. RB1, ERBB2, and CCNE1 alterations were noted among patients with rapid progression on abemaciclib. CONCLUSIONS: A subset of patients with HR+ MBC continue to derive clinical benefit from abemaciclib after progression on prior palbociclib. These results highlight the need for future studies to confirm molecular predictors of cross-resistance to CDK4/6i therapy and to better characterize the utility of abemaciclib after disease progression on prior CDK4/6i.

14.
Nature ; 524(7565): 361-5, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26168401

RESUMO

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy, a conserved self-degradative process. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. Here we show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family of transcription factors. In human PDA cells, the MiT/TFE proteins--MITF, TFE3 and TFEB--are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosome activation is specifically required to maintain intracellular amino acid pools. These results identify the MiT/TFE proteins as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate that transcriptional activation of clearance pathways converging on the lysosome is a novel hallmark of aggressive malignancy.


Assuntos
Autofagia/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica , Lisossomos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metabolismo Energético , Feminino , Xenoenxertos , Homeostase , Humanos , Lisossomos/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Transcrição Gênica
15.
Genes Dev ; 27(2): 182-96, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23322302

RESUMO

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glutamina/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Jejum/metabolismo , Glutationa/biossíntese , Humanos , Larva , Mutação , Nucleotídeos/biossíntese , Estresse Oxidativo , Proteína do Retinoblastoma , Estresse Fisiológico
16.
Nat Rev Mol Cell Biol ; 9(9): 713-24, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18719710

RESUMO

Proteins that are related to the retinoblastoma tumour suppressor pRB and the E2F transcription factor are conserved in many species of plants and animals. The mammalian orthologues of pRB and E2F are best known for their roles in cell proliferation, but it has become clear that they affect many biological processes. Here we describe the functions of pRB-related proteins and E2F proteins that have emerged from genetic and biochemical experiments in Caenorhabditis elegans and Drosophila melanogaster. The similarities that have been observed between worms, flies and mammals provide insight into the core activities of pRB and E2F proteins and show how a common regulatory module can control various biological functions in different organisms.


Assuntos
Fatores de Transcrição E2F/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Apoptose , Linhagem da Célula , Proliferação de Células , Replicação do DNA
17.
Genes Dev ; 26(11): 1128-30, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22661226

RESUMO

In this issue of Genes & Development, Burke and colleagues (pp. 1156-1166) describe how the structure of retinoblastoma protein (pRb) is altered by phosphorylation at T373 or S608. These modifications cause specific conformational changes and alter pRb's interaction with E2F via two distinct mechanisms. The structures suggest that the panel of phosphorylation sites represents a versatile set of tools that are used to sculpt pRb in precise, but very different, ways.


Assuntos
Ciclo Celular , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo
18.
Genes Dev ; 26(4): 356-68, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22345517

RESUMO

E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 3' untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively down-regulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 3' end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio-miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells.


Assuntos
Proteínas de Drosophila/metabolismo , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Drosophila , Proteínas de Drosophila/genética , Fatores de Transcrição E2F/metabolismo , Células HeLa , Humanos , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
19.
Mol Cell ; 42(5): 689-99, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21596603

RESUMO

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Oxirredutases N-Desmetilantes/fisiologia , Receptores Notch/genética , Sirtuína 1/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Imunoprecipitação , Mutação , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fenótipo , Receptores Notch/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
20.
Genes Dev ; 25(8): 814-30, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21498571

RESUMO

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Citometria de Fluxo , Humanos , Immunoblotting , Perda de Heterozigosidade/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Quinases Dyrk
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