RESUMEN
The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Calreticulina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/metabolismo , Empalme Alternativo/genética , Factor de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Calreticulina/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/patología , Chaperón BiP del Retículo Endoplásmico , Regulación Leucémica de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Células Mieloides/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Biosíntesis de Proteínas , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional/genética , Proteína 1 de Unión a la X-Box , Factor de Transcripción YY1/metabolismoRESUMEN
PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Pliegue de Proteína , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo , Proteínas Potenciadoras de Unión a CCAAT/genética , Calreticulina/genética , Aberraciones Cromosómicas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/genética , Humanos , Cariotipificación , Leucemia Mieloide/metabolismo , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/genética , Mutación , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Factor de Transcripción CHOP/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , Adulto JovenRESUMEN
microRNA-223 (miR-223) can trigger normal granulopoiesis. miR-223 expression is regulated by two distinct CEBPA (CCAAT/enhancer binding protein-alpha) sites. Here, we report that miR-223 is largely suppressed in cells from acute myeloid leukemia (AML) patients. By sequencing, we found that miR-223 suppression in AML is not caused by DNA sequence alterations, nor is it mediated by promoter hypermethylation. The analysis of the individual contribution of both CEBPA sites to miR-223 regulation identified the site upstream of the miR-223 primary transcript as the predominant regulatory element. Our results suggest that miR-223 suppression in AML is caused by impaired miR-223 upstream factors.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Regulación Leucémica de la Expresión Génica/genética , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Secuencia de Bases , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all-trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Antineoplásicos/farmacología , Western Blotting , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Diferenciación Celular/fisiología , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Macrófagos/citología , Macrófagos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacología , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: The transcription factor CCAAT/enhancer binding protein-alpha (CEBPA) is crucial for normal myeloid differentiation. Mutations in the CEBPA gene are found in subsets of patients with acute myeloid leukemia (AML). Recently, three families were reported in whom several family members had germline CEBPA mutations and subsequently developed AML. Whereas familial AML is considered a rare event, the frequency of CEBPA germline mutations in AML is not known. PATIENTS AND METHODS: In this study, we screened 187 consecutive AML patients for CEBPA mutations at diagnosis. We detected 18 patients (9.6%) with CEBPA mutations. We then analyzed remission samples and constitutive DNA from these patients. RESULTS: We found that two (11.1%) of 18 AML patients with CEBPA mutations carried a germline N-terminal frameshift CEBPA mutation. Interestingly, additional members in the families of both of these patients have been affected by AML, and the germline CEBPA mutations were also observed in these patients. Additional somatic mutations in AML patients with germline CEBPA mutations in the two families comprised in-frame C-terminal CEBPA mutations in two patients, two nonsilent CEBPA point mutations in one patient, and monosomy 7 in one patient. CONCLUSION: This study shows, for the first time to our knowledge, that germline CEBPA mutations are frequently observed among AML patients with CEBPA mutations. Including the families with germline CEBPA mutations reported previously, additional somatic CEBPA mutations represent a frequent second event in AML with germline CEBPA mutations. Our data strongly indicate that germline CEBPA mutations predispose to AML and that additional somatic CEBPA mutations contribute to the development of the disease.