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1.
Nature ; 494(7438): 492-496, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23446422

RESUMO

Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.


Assuntos
Instabilidade Cromossômica/genética , Neoplasias Colorretais/genética , Replicação do DNA/genética , Aneuploidia , Linhagem Celular Tumoral , Instabilidade Cromossômica/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cromossomos Humanos Par 18/efeitos dos fármacos , Cromossomos Humanos Par 18/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA/efeitos dos fármacos , Deleção de Genes , Inativação Gênica , Genes Supressores de Tumor , Humanos , Mitose/efeitos dos fármacos , Nucleosídeos/farmacologia , Fosfotransferases/genética , Proteínas de Ligação a RNA/genética
2.
Nature ; 460(7251): 123-7, 2009 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-19483676

RESUMO

Apoptosis is a conserved form of programmed cell death firmly established in the aetiology, pathogenesis and treatment of many human diseases. Central to the core machinery of apoptosis are the caspases and their proximal regulators. Current models for caspase control involve a balance of opposing elements, with variable contributions from positive and negative regulators among different cell types and species. To advance a comprehensive view of components that support caspase-dependent cell death, we conducted a genome-wide silencing screen in the Drosophila model. Our strategy used a library of double-stranded RNAs together with a chemical antagonist of Inhibitor of apoptosis proteins (IAPs) that simulates the action of native regulators in the Reaper and Smac (also known as Diablo) families. Here we present a highly validated set of targets that is necessary for death provoked by several stimuli. Among these, Tango7 is identified as a new effector. Cells depleted for this gene resisted apoptosis at a step before the induction of effector caspase activity, and the directed silencing of Tango7 in Drosophila prevented caspase-dependent programmed cell death. Unlike known apoptosis regulators in this model system, Tango7 activity did not influence stimulus-dependent loss of Drosophila DIAP1 (also known as th and IAP1), but instead regulated levels of the apical caspase Dronc (Nc). Similarly, the human Tango7 counterpart, PCID1 (also known as EIF3M), impinged on caspase 9, revealing a new regulatory axis affecting the apoptosome.


Assuntos
Apoptose/genética , Apoptose/fisiologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fatores de Iniciação em Eucariotos/metabolismo , Inativação Gênica , Genoma de Inseto/genética , Animais , Apoptossomas/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Caspase 9/metabolismo , Caspases/metabolismo , Sequência Conservada , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Fator de Iniciação 3 em Eucariotos , Genes de Insetos/genética , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais , Mimetismo Molecular , Interferência de RNA , RNA de Cadeia Dupla/genética , Reprodutibilidade dos Testes , Proteínas de Xenopus
3.
Am J Pathol ; 182(6): 1962-71, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23708210

RESUMO

Emerging evidence suggests that cancer branched evolution may affect biomarker validation, clinical outcome, and emergence of drug resistance. The changing spatial and temporal nature of cancer subclonal architecture during the disease course suggests the need for longitudinal prospective studies of cancer evolution and robust and clinically implementable pathologic definitions of intratumor heterogeneity, genetic diversity, and chromosomal instability. Furthermore, subclonal heterogeneous events in tumors may evade detection through conventional biomarker strategies and influence clinical outcome. Minimally invasive methods for the study of cancer evolution and new approaches to clinical study design, incorporating understanding of the dynamics of tumor clonal architectures through treatment and during acquisition of drug resistance, have been suggested as important areas for development. Coordinated efforts will be required by the scientific and clinical trial communities to adapt to the challenges of detecting infrequently occurring somatic events that may influence clinical outcome and to understand the dynamics of cancer evolution and the waxing and waning of tumor subclones over time in advanced metastatic epithelial malignancies.


Assuntos
Evolução Clonal , Neoplasias/genética , Biomarcadores Tumorais/metabolismo , Variação Genética , Humanos , Mutação , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/patologia , Filogenia , Projetos de Pesquisa , Microambiente Tumoral/genética
4.
Nat Commun ; 15(1): 4871, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38871738

RESUMO

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.


Assuntos
Instabilidade Cromossômica , Receptores ErbB , Neoplasias Pulmonares , Mutação , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Terapia de Alvo Molecular/métodos , Feminino , Variações do Número de Cópias de DNA , Masculino
5.
Methods ; 53(4): 366-71, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21185377

RESUMO

Transposons are an attractive system to use in genetic screens as they are molecularly tractable and the disrupted loci that give rise to the desired phenotype are easily mapped. We consider herein the characteristics of the piggyBac transposon system in complementing existing mammalian screen strategies, including the Sleeping Beauty transposon system. We also describe the design of the piggyBac resources that we have developed for both forward and reverse genetic screens, and the protocols we use in these experiments.


Assuntos
Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis , Animais , Técnicas de Cocultura , Células-Tronco Embrionárias , Componentes do Gene , Genes Neoplásicos , Vetores Genéticos , Humanos , Hibridização Genética , Estimativa de Kaplan-Meier , Camundongos , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Transfecção/métodos , Transposases/genética
6.
Cancer Res ; 82(8): 1589-1602, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35425962

RESUMO

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Animais , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Oncogenes , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
Genesis ; 49(8): 689-95, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21786403

RESUMO

Cre-loxP recombination is widely used for genetic manipulation of the mouse genome. Here, we report generation and characterization of a new Cre line, Stella-Cre, where Cre expression cassette was targeted to the 3' UTR of the Stella locus. Stella is specifically expressed in preimplantation embryos and in the germline. Cre-loxP recombination efficiency in Stella-Cre mice was investigated at several genomic loci including Rosa26, Jak2, and Npm1. At all the loci examined, we observed 100% Cre-loxP recombination efficiency in the embryos and in the germline. Thus, Stella-Cre mice serve as a very efficient deleter line.


Assuntos
Expressão Gênica , Integrases/genética , Recombinação Genética , Proteínas Repressoras/genética , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação Microbiológicos/genética , Bacteriófago P1/genética , Blastocisto , Proteínas Cromossômicas não Histona , Embrião de Mamíferos/metabolismo , Feminino , Citometria de Fluxo , Células Germinativas/metabolismo , Integrases/metabolismo , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Proteínas Nucleares/genética , Nucleofosmina , Proteínas/genética , RNA não Traduzido , Proteínas Repressoras/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
8.
Cancer Discov ; 11(7): 1754-1773, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33608386

RESUMO

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.


Assuntos
Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transformação Celular Neoplásica , Humanos , Neoplasias Pulmonares/patologia
9.
Dev Cell ; 7(6): 897-907, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15572131

RESUMO

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis.


Assuntos
Apoptose , Caspases/fisiologia , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Alelos , Animais , Padronização Corporal , Caspases/metabolismo , Morte Celular , Drosophila melanogaster , Olho/embriologia , Olho/metabolismo , Teste de Complementação Genética , Genoma , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/metabolismo , Homozigoto , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Modelos Genéticos , Mutagênese , Mutação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
10.
Nat Commun ; 8(1): 2026, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229903

RESUMO

Inactivation of the VHL (Von Hippel Lindau) tumour suppressor has long been recognised as necessary for the pathogenesis of clear cell renal cancer (ccRCC); however, the molecular mechanisms underlying transformation and the requirement for additional genetic hits remain unclear. Here, we show that loss of VHL alone results in DNA replication stress and damage accumulation, effects that constrain cellular growth and transformation. By contrast, concomitant loss of the chromatin remodelling factor PBRM1 (mutated in 40% of ccRCC) rescues VHL-induced replication stress, maintaining cellular fitness and allowing proliferation. In line with these data we demonstrate that combined deletion of Vhl and Pbrm1 in the mouse kidney is sufficient for the development of fully-penetrant, multifocal carcinomas, closely mimicking human ccRCC. Our results illustrate how VHL and PBRM1 co-operate to drive renal transformation and uncover replication stress as an underlying vulnerability of all VHL mutated renal cancers that could be therapeutically exploited.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas HMGB/genética , Rim/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas HMGB/metabolismo , Humanos , Estimativa de Kaplan-Meier , Rim/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
11.
Genome Biol ; 15(9): 455, 2014 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-25260652

RESUMO

The in vivo validation of cancer mutations and genes identified in cancer genomics is resource-intensive because of the low throughput of animal experiments. We describe a mouse model that allows multiple cancer mutations to be validated in each animal line. Animal lines are generated with multiple candidate cancer mutations using transposons. The candidate cancer genes are tagged and randomly expressed in somatic cells, allowing easy identification of the cancer genes involved in the generated tumours. This system presents a useful, generalised and efficient means for animal validation of cancer genes.


Assuntos
Estudos de Associação Genética/métodos , Neoplasias/genética , Animais , Carcinogênese/genética , Células Cultivadas , Técnicas de Cocultura , Elementos de DNA Transponíveis , Predisposição Genética para Doença , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Herança Multifatorial , Mutação , Transplante de Neoplasias
12.
Plant J ; 34(5): 573-83, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12787240

RESUMO

The key role for mitochondria in mammalian apoptosis, a form of programmed cell death (PCD), is well established, but a similar role for plant mitochondria is just emerging. In order to unravel the molecular mechanisms linking plant mitochondria to the downstream events of PCD, we have developed an Arabidopsis cell-free system that can be used to monitor biochemical and morphological changes in isolated nuclei that are associated with PCD. Using this system, two activities that resulted in nuclear DNA degradation could be distinguished, both of which were facilitated by the addition of mitochondria. One activity mediated the generation of 30 kb DNA fragments within 3 h and chromatin condensation within 6 h, when nuclei were incubated with mitochondria alone. The second activity required cytosolic extract in addition to mitochondria and resulted in oligonucleosome-sized DNA cleavage after >12 h. Submitochondrial fractionation and pharmacological studies suggested the presence of an Mg2+-dependent nuclease activity in the intermembrane space, which is responsible for the former in vitro activity. The evolutionary conservation of the role of mitochondria in PCD in animals and plants is discussed.


Assuntos
Apoptose , Arabidopsis/citologia , Arabidopsis/enzimologia , Desoxirribonucleases/metabolismo , Mitocôndrias/enzimologia , Extratos Celulares , Núcleo Celular/metabolismo , DNA/química , DNA/metabolismo , Fragmentação do DNA , Temperatura Alta , Dados de Sequência Molecular
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