RESUMEN
Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.
Asunto(s)
Proteínas de Unión al ADN/genética , Granulocitos/citología , Hematopoyesis/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Neutropenia/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Mutación , Neutropenia/congénito , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
K-RAS mutations are found in approximately 30% of lung cancers. The transcription factor Krüppel-like Factor 5 (KLF5) has been shown to mediate cellular transformation signaling events downstream of oncogenic RAS in other cancers, but a role for KLF5 in lung tumorigenesis has not been defined. We show here that knockdown of KLF5 expression significantly decreased anchorage-independent growth, but did not affect proliferation of human lung adenocarcinoma cells. Moreover, Klf5 is not required for lung tumor formation in an inducible oncogenic K-Ras(G12D) mouse model of lung tumorigenesis, and non-small cell lung cancer patients expressing high levels of KLF5 (21/258) have a significantly better disease-specific survival than those with intermediate to no KLF5 expression. Further, KLF5 knockdown in K-RAS-mutant human lung cancer cells resulted in a fivefold increase in ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2), an anthracycline drug transporter, which lead to significantly increased resistance to doxorubicin treatment, a chemotherapeutic agent clinically used to treat lung cancer. In summary, while KLF5 is not required for oncogenic mutant K-Ras-induced lung tumorigenesis, KLF5 regulation of ABCG2 expression may be important for chemotherapeutic resistance and patient survival.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Análisis de Varianza , Animales , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Supervivencia sin Enfermedad , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Matrices Tisulares , Proteínas ras/genéticaRESUMEN
The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs, and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically, bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However, coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus, our results not only identify microRNAs whose regulation is required during myelopoiesis, but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis.