RESUMO
Ferroptosis is a specialized iron-dependent cell death that is associated with lethal lipid peroxidation. Modulation of ferroptosis may have therapeutic potential since it has been implicated in various human diseases as well as potential antitumor activities. However, much remains unknown about the underlying mechanisms and genetic determinants of ferroptosis. Given the critical role of kinases in most biological processes and the availability of various kinase inhibitors, we sought to systemically identify kinases essential for ferroptosis. We performed a forward genetic-based kinome screen against ferroptosis in MDA-MB-231 cells triggered by cystine deprivation. This screen identified 34 essential kinases involved in TNFα and NF-kB signaling. Unexpectedly, the DNA damage response serine/threonine kinase ATM (mutated in Ataxia-Telangiectasia) was found to be essential for ferroptosis. The pharmacological or genetic inhibition of ATM consistently rescued multiple cancer cells from ferroptosis triggered by cystine deprivation or erastin. Instead of the canonical DNA damage pathways, ATM inhibition rescued ferroptosis by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chain, FTH1 and FTL) and export (ferroportin, FPN1). The coordinated changes of these iron regulators during ATM inhibition resulted in a lowering of labile iron and prevented the iron-dependent ferroptosis. Furthermore, we found that ATM inhibition enhanced the nuclear translocation of metal-regulatory transcription factor 1 (MTF1), responsible for regulating expression of Ferritin/FPN1 and ferroptosis protection. Genetic depletion of MTF-1 abolished the regulation of iron-regulatory elements by ATM and resensitized the cells to ferroptosis. Together, we have identified an unexpected ATM-MTF1-Ferritin/FPN1 regulatory axis as novel determinants of ferroptosis through regulating labile iron levels.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ferroptose , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Cistina/metabolismo , Ferroptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ferro/metabolismo , Modelos Biológicos , Piperazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The temporal activation of kinases and timely ubiquitin-mediated degradation is central to faithful mitosis. Here we present evidence that acetylation controlled by Coenzyme A synthase (COASY) and acetyltransferase CBP constitutes a novel mechanism that ensures faithful mitosis. We found that COASY knockdown triggers prolonged mitosis and multinucleation. Acetylome analysis reveals that COASY inactivation leads to hyper-acetylation of proteins associated with mitosis, including CBP and an Aurora A kinase activator, TPX2. During early mitosis, a transient CBP-mediated TPX2 acetylation is associated with TPX2 accumulation and Aurora A activation. The recruitment of COASY inhibits CBP-mediated TPX2 acetylation, promoting TPX2 degradation for mitotic exit. Consistently, we detected a stage-specific COASY-CBP-TPX2 association during mitosis. Remarkably, pharmacological and genetic inactivation of CBP effectively rescued the mitotic defects caused by COASY knockdown. Together, our findings uncover a novel mitotic regulation wherein COASY and CBP coordinate an acetylation network to enforce productive mitosis.
Assuntos
Proteína de Ligação a CREB/metabolismo , Mitose , Transferases/metabolismo , Acetilação , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteína de Ligação a CREB/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Transferases/genéticaRESUMO
A body of evidence has shown that the control of E2F transcription factor activity is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell-cycle regulatory activities, has not been clearly defined. Here, recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we model the potential roles of individual components together with the use of an integrated system to follow E2F dynamics at the single-cell level and in real time. These analyses reveal that crossing a threshold amplitude of E2F accumulation determines cell cycle commitment. Importantly, we find that Myc is critical in modulating the amplitude, whereas cyclin D/E activities have little effect on amplitude but do contribute to the modulation of duration of E2F activation, thereby affecting the pace of cell cycle progression.
Assuntos
Ciclo Celular/genética , Ciclina D/genética , Ciclina E/genética , Fator de Transcrição E2F1/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Ciclina D/metabolismo , Ciclina E/metabolismo , Fator de Transcrição E2F1/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células NIH 3T3 , Piperazinas/farmacologia , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Purinas/farmacologia , Piridinas/farmacologia , Ratos , Transdução de Sinais , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
Gene expression signatures can predict the activation of oncogenic pathways and other phenotypes of interest via quantitative models that combine the expression levels of multiple genes. However, as the number of platforms to measure genome-wide gene expression proliferates, there is an increasing need to develop models that can be ported across diverse platforms. Because of the range of technologies that measure gene expression, the resulting signal values can vary greatly. To understand how this variation can affect the prediction of gene expression signatures, we have investigated the ability of gene expression signatures to predict pathway activation across Affymetrix and Illumina microarrays. We hybridized the same RNA samples to both platforms and compared the resultant gene expression readings, as well as the signature predictions. Using a new approach to map probes across platforms, we found that the genes in the signatures from the two platforms were highly similar, and that the predictions they generated were also strongly correlated. This demonstrates that our method can map probes from Affymetrix and Illumina microarrays, and that this mapping can be used to predict gene expression signatures across platforms.
Assuntos
Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular Tumoral , Estudo de Associação Genômica Ampla , HumanosRESUMO
Stimulation of quiescent mammalian cells with mitogens induces an abrupt increase in E2F1-3 expression just prior to the onset of DNA synthesis, followed by a rapid decline as replication ceases. This temporal adaptation in E2F facilitates a transient pattern of gene expression that reflects the ordered nature of DNA replication. The challenge to understand how E2F dynamics coordinate molecular events required for high-fidelity DNA replication has great biological implications. Indeed, precocious, prolonged, elevated or reduced accumulation of E2F can generate replication stress that culminates in either arrest or death. Accordingly, temporal characteristics of E2F are regulated by several network modules that include feedforward and autoregulatory loops. In this review, we discuss how these network modules contribute to "shaping" E2F dynamics in the context of mammalian cell cycle entry.
Assuntos
Ciclo Celular , Fatores de Transcrição E2F/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Dano ao DNA , Replicação do DNA , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mamíferos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Origem de Replicação , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
The hallmark of human cancer is heterogeneity, reflecting the complexity and variability of the vast array of somatic mutations acquired during oncogenesis. An ability to dissect this heterogeneity, to identify subgroups that represent common mechanisms of disease, will be critical to understanding the complexities of genetic alterations and to provide a framework to develop rational therapeutic strategies. Here, we describe a classification scheme for human breast cancer making use of patterns of pathway activity to build on previous subtype characterizations using intrinsic gene expression signatures, to provide a functional interpretation of the gene expression data that can be linked to therapeutic options. We show that the identified subgroups provide a robust mechanism for classifying independent samples, identifying tumors that share patterns of pathway activity and exhibit similar clinical and biological properties, including distinct patterns of chromosomal alterations that were not evident in the heterogeneous total population of tumors. We propose that this classification scheme provides a basis for understanding the complex mechanisms of oncogenesis that give rise to these tumors and to identify rational opportunities for combination therapies.
Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/diagnóstico , Regulação Neoplásica da Expressão Gênica , Algoritmos , Linhagem Celular Tumoral , Análise por Conglomerados , DNA/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Genômica , Humanos , Modelos Genéticos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos/genética , FenótipoRESUMO
Tetraspanins are evolutionary conserved transmembrane proteins thought to facilitate cell proliferation, movement or fusion by acting as organizers of different signaling events. Despite their prevalence and conservation, their specific role and functions remain largely elusive, as their redundancy in various organisms has hindered loss of function studies. Here, we take a gain of function approach to study Drosophila tetraspanin Tsp68C and its effect on larval hemocytes. We recently characterized a lethal mutation in ytr, a conserved gene that encodes a nuclear arginine-rich protein of unknown function, which is accompanied by abnormal differentiation and proliferation of the larval hematopoietic tissue in flies. A hemolectin (hml)-Gal4 construct carried by hml-Gal4 transgenic flies was sufficient by itself to abrogate the hematopoietic defects in ytr mutant larvae. This rescue correlated with the overexpression of tsp68C, a tetraspanin gene nested in the hml promoter. The suppression of abnormal proliferation by the hml-Gal4 construct was not restricted to ytr-deficient hemocytes, but was also observed in hemocytes expressing the oncogenic forms of Raf or Ras proteins. However, it had no effect on overproliferation mediated by a constitutively active form of Jak. New hml-Gal4 lines, in which the tsp68C gene was silenced or deleted from the promoter, no longer rescued the hematopoietic defect in ytr mutants nor suppressed the activated Raf-induced overproliferation. Therefore, change in tetraspanin Tsp68C expression has a strong suppressor effect on abnormal proliferation and differentiation of hemocytes in the context of specific lesions, such as overactivation of the Ras/Raf/MAPK pathway.
Assuntos
Divisão Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Hemócitos/fisiologia , Proteínas de Membrana/genética , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Quinases raf/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA , Drosophila , Proteínas de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Hematopoese/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Northern Blotting , Células COS , Chlorocebus aethiops , Mapeamento Cromossômico , Primers do DNA , Drosophila/metabolismo , Hemócitos/citologia , Imuno-Histoquímica , Hibridização In Situ , Larva/metabolismo , Larva/fisiologia , Camundongos , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in Drosophila. Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Sistema de Sinalização das MAP Quinases/genética , Mutação , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Sequência de Bases , Sequência Consenso , Drosophila melanogaster/embriologia , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Plasmídeos , Reação em Cadeia da Polimerase , Fatores de Transcrição STATRESUMO
We have cloned a novel SOCS gene from Drosophila, socs36E, which is most homologous to the mammalian socs-5 gene. Socs36E is expressed zygotically, predominantly during embryogenesis, in a highly dynamic pattern. In vivo expression of SOCS36E in transgenic flies results in several adult phenotypes. Engrailed-GAL4 directed expression causes loss of the wing anterior cross vein, humeral outgrowths, absence of halteres and eye pigmentation defects. Expression of SOCS36E under apterous-GAL4 control resulted in outstretched wings. Full penetrance of these phenotypes required the presence of the SH2 and SOCS-box domains of SOCS36E. The observed phenotypes were consistent with defects in JAK/STAT or EGF-R signalling and were exacerbated in flies heterozygous for either the d-jak (hopscotch), d-stat (stat92E) or d-egf-r (der) genes. Conversely, inactivating one copy of the d-cbl gene, a negative regulator of the d-EGF-R, partially rescued the wing phenotypes. These genetic interactions imply that SOCS36E can suppress activities of the JAK/STAT and EGF-R signalling pathways in the wing disc and suggest that SOCS36E interacts with multiple pathways in vivo.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Drosophila/metabolismo , Proteínas Quinases , Proteínas Repressoras/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/antagonistas & inibidores , Drosophila/genética , Proteínas de Drosophila/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Janus Quinases , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Peptídeos de Invertebrados/antagonistas & inibidores , Proteínas Repressoras/genética , Fatores de Transcrição STAT , Alinhamento de Sequência , Proteínas Supressoras da Sinalização de Citocina , Transativadores/antagonistas & inibidores , Fatores de Transcrição , Asas de Animais/anatomia & histologia , Asas de Animais/embriologia , Asas de Animais/metabolismoRESUMO
The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.