RESUMO
The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.
Assuntos
Técnicas Genéticas , Vetores Genéticos/genética , Lentivirus/genética , Interferência de RNA , Animais , Neoplasias da Mama/patologia , Linhagem Celular , DNA Complementar/genética , Diagnóstico por Imagem , Feminino , Expressão Gênica , Humanos , Luminescência , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lineage selective transcription factors (TFs) are important regulators of tumorigenesis, but their biological functions are often context dependent with undefined epigenetic mechanisms of action. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 in lung adenocarcinoma (LUAD) progression. Impairing Gata6 in genetically engineered mouse models reduces the proliferation and increases the differentiation of Kras mutant LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation and genetic context of Kras-mediated tumor initiation. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in lung development, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse transcriptional programs associated with bone morphogenetic protein signaling, alveolar specification, and tumor suppression. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their proliferation and divergent lineage dependencies during tumor progression.
Assuntos
Adenocarcinoma de Pulmão/genética , Fator de Transcrição GATA6/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/patologia , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Cromatina/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , CamundongosRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.
Assuntos
Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Aminopiridinas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Neoplasias Cerebelares/tratamento farmacológico , Cerebelo/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genes p53/fisiologia , Imidazóis/farmacologia , Meduloblastoma/tratamento farmacológico , Camundongos , Morfolinas/farmacologia , Células-Tronco Neurais/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/fisiologiaRESUMO
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.