RESUMEN
The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell acute lymphoblastic leukemia (B-ALL) associated with reduced expression of the established B-lineage tumor suppressor genes, Ikaros and Spi-B. These genes are directly regulated by PU.1/IRF4/IRF8, and restoration of Ikaros or Spi-B expression inhibited leukemic cell growth. In summary, we demonstrate that PU.1, IRF4 and IRF8 cooperate to regulate early B-cell development and to prevent pre-B-ALL formation.
Asunto(s)
Factores Reguladores del Interferón/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Linfocitos B/citología , Regulación de la Expresión Génica , Factores Reguladores del Interferón/genética , Linfopoyesis , Ratones , Ratones Noqueados , Leucemia-Linfoma Linfoblástico de Células Precursoras B/prevención & control , Proteínas Proto-Oncogénicas/genética , Transactivadores/genéticaRESUMEN
Activating NOTCH1 mutations occur in ~60% of human T-cell acute lymphoblastic leukemias (T-ALLs), and mutations disrupting the transcription factor IKZF1 (IKAROS) occur in ~5% of cases. To investigate the regulatory interplay between these driver genes, we have used a novel transgenic RNA interference mouse model to produce primary T-ALLs driven by reversible Ikaros knockdown. Restoring endogenous Ikaros expression in established T-ALL in vivo acutely represses Notch1 and its oncogenic target genes including Myc, and in multiple primary leukemias causes disease regression. In contrast, leukemias expressing high levels of endogenous or engineered forms of activated intracellular Notch1 (ICN1) resembling those found in human T-ALL rapidly relapse following Ikaros restoration, indicating that ICN1 functionally antagonizes Ikaros in established disease. Furthermore, we find that IKAROS mRNA expression is significantly reduced in a cohort of primary human T-ALL patient samples with activating NOTCH1/FBXW7 mutations, but is upregulated upon acute inhibition of aberrant NOTCH signaling across a panel of human T-ALL cell lines. These results demonstrate for the first time that aberrant NOTCH activity compromises IKAROS function in mouse and human T-ALL, and provide a potential explanation for the relative infrequency of IKAROS gene mutations in human T-ALL.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Factor de Transcripción Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Biomarcadores de Tumor/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Inmunoprecipitación de Cromatina , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factor de Transcripción Ikaros/antagonistas & inhibidores , Factor de Transcripción Ikaros/genética , Ratones , Ratones Transgénicos , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Notch/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1-34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Diferenciación Celular/genética , Proliferación Celular/genética , Osteosarcoma/genética , Osteosarcoma/patología , Receptor de Hormona Paratiroídea Tipo 1/genética , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , ARN Interferente Pequeño/farmacología , Receptor de Hormona Paratiroídea Tipo 1/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
Evasion of cell death is fundamental to the development of cancer and its metastasis. The role of the BCL-2-mediated (intrinsic) apoptotic program in these processes remains poorly understood. Here we have investigated the relevance of the pro-apoptotic protein BIM to breast cancer progression using the MMTV-Polyoma middle-T (PyMT) transgenic model. BIM deficiency in PyMT females did not affect primary tumor growth, but substantially increased the survival of metastatic cells within the lung. These data reveal a role for BIM in the suppression of breast cancer metastasis. Intriguingly, we observed a striking correlation between the expression of BIM and the epithelial to mesenchymal transition transcription factor SNAI2 at the proliferative edge of the tumors. Overexpression and knockdown studies confirmed that these two genes were coordinately expressed, and chromatin immunoprecipitation analysis further revealed that Bim is a target of SNAI2. Taken together, our findings suggest that SNAI2-driven BIM-induced apoptosis may temper metastasis by governing the survival of disseminating breast tumor cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/fisiología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Supervivencia Celular , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción de la Familia Snail , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. OBJECTIVE: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. METHODS AND RESULTS: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L) , a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L) , clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. CONCLUSIONS: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.