RESUMEN
Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3ß. While STAT3α is predominantly described as an oncogenic driver, STAT3ß has been suggested to act as a tumor suppressor. To elucidate the role of STAT3ß in AML, we established a mouse model of STAT3ß-deficient, MLL-AF9-driven AML. STAT3ß deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3ß. Accordingly, STAT3ß-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3ß expression. Together, our data corroborate the tumor suppressive role of STAT3ß in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3ß/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3ß/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.
Asunto(s)
Leucemia Mieloide Aguda , Factor de Transcripción STAT3 , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Humanos , Factor de Transcripción STAT3/metabolismo , Ratones , Transducción de Señal , Interferones/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/genética , Línea Celular Tumoral , Nitrilos , Pirazoles , PirimidinasRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide. The main risk factors are consumption of tobacco products and alcohol, as well as infection with human papilloma virus. Approved therapeutic options comprise surgery, radiation, chemotherapy, targeted therapy through epidermal growth factor receptor inhibition, and immunotherapy, but outcome has remained unsatisfactory due to recurrence rates of ~50% and the frequent occurrence of second primaries. The availability of the human genome sequence at the beginning of the millennium heralded the omics era, in which rapid technological progress has advanced our knowledge of the molecular biology of malignant diseases, including HNSCC, at an unprecedented pace. Initially, microarray-based methods, followed by approaches based on next-generation sequencing, were applied to study the genetics, epigenetics, and gene expression patterns of bulk tumors. More recently, the advent of single-cell RNA sequencing (scRNAseq) and spatial transcriptomics methods has facilitated the investigation of the heterogeneity between and within different cell populations in the tumor microenvironment (e.g., cancer cells, fibroblasts, immune cells, endothelial cells), led to the discovery of novel cell types, and advanced the discovery of cell-cell communication within tumors. This review provides an overview of scRNAseq, spatial transcriptomics, and the associated bioinformatics methods, and summarizes how their application has promoted our understanding of the emergence, composition, progression, and therapy responsiveness of, and intercellular signaling within, HNSCC.
Asunto(s)
Células Endoteliales , Neoplasias de Cabeza y Cuello , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Perfilación de la Expresión Génica , Biología Computacional , Progresión de la Enfermedad , Análisis de Secuencia de ARN , Microambiente Tumoral/genéticaRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor prognosis. So far, the EGFR inhibitor cetuximab is the only approved targeted therapy. A deeper understanding of the molecular and genetic basis of HNSCC is needed to identify additional targets for rationally designed, personalized therapeutics. The transcription factor EVI1, the major product of the MECOM locus, is an oncoprotein with roles in both hematological and solid tumors. In HNSCC, high EVI1 expression was associated with an increased propensity to form lymph node metastases, but its effects in this tumor entity have not yet been determined experimentally. We therefore overexpressed or knocked down EVI1 in several HNSCC cell lines and determined the impact of these manipulations on parameters relevant to tumor growth and invasiveness, and on gene expression patterns. Our results revealed that EVI1 promoted the proliferation and migration of HNSCC cells. Furthermore, it augmented tumor spheroid formation and the ability of tumor spheroids to displace an endothelial cell layer. Finally, EVI1 altered the expression of numerous genes in HNSCC cells, which were enriched for Gene Ontology terms related to its cellular functions. In summary, EVI1 represents a novel oncogene in HNSCC that contributes to cellular proliferation and invasiveness.
Asunto(s)
Neoplasias de Cabeza y Cuello , Proteína del Locus del Complejo MDS1 y EV11 , Carcinoma de Células Escamosas de Cabeza y Cuello , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Proteína del Locus del Complejo MDS1 y EV11/genética , Invasividad Neoplásica , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Factores de Transcripción/genéticaRESUMEN
Relapse is associated with therapy resistance and is a major cause of death in acute myeloid leukemia (AML). It is thought to result from the accretion of therapy-refractory leukemic stem cells. Genetic and transcriptional changes that are recurrently gained at relapse are likely to contribute to the increased stemness and decreased therapy responsiveness at this disease stage. Despite the recent approval of several targeted drugs, chemotherapy with cytosine arabinoside and anthracyclines is still the mainstay of AML therapy. Accordingly, a number of studies have investigated genetic and gene expression changes between diagnosis and relapse of patients subjected to such treatment. Genetic alterations recurrently acquired at relapse were identified, but were restricted to small proportions of patients, and their functional characterization is still largely pending. In contrast, the expression of a substantial number of genes was altered consistently between diagnosis and recurrence of AML. Recent studies corroborated the roles of the upregulation of SOCS2 and CALCRL and of the downregulation of MTSS1 and KDM6A in therapy resistance and/or stemness of AML. These findings spur the assumption that functional investigations of genes consistently altered at recurrence of AML have the potential to promote the development of novel targeted drugs that may help to improve the outcome of this currently often fatal disease.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/biosíntesis , Regulación Leucémica de la Expresión Génica , Histona Demetilasas/biosíntesis , Leucemia Mieloide Aguda , Proteínas de Microfilamentos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , RecurrenciaRESUMEN
Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/patología , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Antraciclinas/administración & dosificación , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Neoplasias/genética , Pronóstico , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Tasa de SupervivenciaRESUMEN
All-trans retinoic acid (atRA) has a dramatic impact on the survival of patients with acute promyelocytic leukemia, but its therapeutic value in other types of acute myeloid leukemia (AML) has so far remained unclear. Given that AML is a stem cell-driven disease, recent studies have addressed the effects of atRA on leukemic stem cells (LSCs). atRA promoted stemness of MLL-AF9-driven AML in an Evi1-dependent manner but had the opposite effect in Flt3-ITD/Nup98-Hoxd13-driven AML. Overexpression of the stem cell-associated transcription factor EVI1 predicts a poor prognosis in AML, and is observed in different genetic subtypes, including cytogenetically normal AML. Here, we therefore investigated the effects of Evi1 in a mouse model for cytogenetically normal AML, which rests on the combined activity of Flt3-ITD and Npm1c mutations. Experimental expression of Evi1 on this background strongly promoted disease aggressiveness. atRA inhibited leukemia cell viability and stem cell-related properties, and these effects were counteracted by overexpression of Evi1. These data further underscore the complexity of the responsiveness of AML LSCs to atRA and point out the need for additional investigations which may lay a foundation for a precision medicine-based use of retinoids in AML.
RESUMEN
Overexpression of IL2RA, which encodes the alpha chain of the IL2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia (AML). The clinical potential of anti-IL2RA therapy is, therefore, being explored in early-stage clinical trials. Notwithstanding, only very limited information regarding the biological function of IL2RA in AML is available. Using genetic manipulation of IL2RA expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of IL2RA on AML cell proliferation and apoptosis, and on pertinent signaling pathways. The impact of IL2RA on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. IL2RA promoted proliferation and cell-cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell-cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of IL2RA were confirmed in two genetically distinct AML mouse models, revealing that IL2RA inhibits differentiation, promotes stem cell-related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergized with other antileukemic agents in this regard. Collectively, these data show for the first time that IL2RA plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease. SIGNIFICANCE: This study identifies IL2RA as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell-related properties, and leukemogenesis.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Ratones Endogámicos C57BL , Pronóstico , Células Madre/patología , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Acute myeloid leukemia (AML) is an aggressive, often fatal hematopoietic malignancy. All-trans retinoic acid (atRA), one of the first molecularly targeted drugs in oncology, has greatly improved the outcome of a subtype of AML, acute promyelocytic leukemia (APL). In contrast, atRA has so far provided little therapeutic benefit in the much larger group of patients with non-APL AML. Attempts to identify genetically or molecularly defined subgroups of patients that may respond to atRA have not yielded consistent results. Since AML is a stem cell-driven disease, understanding the effectiveness of atRA may require an appreciation of its impact on AML stem cells. Recent studies reported that atRA decreased stemness of AML with an FLT3-ITD mutation, yet increased it in AML1-ETO driven or EVI1-overexpressing AML. This review summarizes the role of atRA in normal hematopoiesis and in AML, focusing on its impact on AML stem cells.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Tretinoina/farmacología , Animales , Apoptosis/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , HumanosRESUMEN
The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3'UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.
Asunto(s)
Proteína del Locus del Complejo MDS1 y EV11/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/genética , Regiones no Traducidas 3' , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Carcinoma Nasofaríngeo/patología , Células Madre Neoplásicas/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteína del Locus del Complejo MDS1 y EV11/genética , Receptor Notch4/genética , Tretinoina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Células Mieloides/efectos de los fármacos , Células Madre Neoplásicas/metabolismoRESUMEN
The neuropeptide CGRP, acting through the G-protein coupled receptor CALCRL and its coreceptor RAMP1, plays a key role in migraines, which has led to the clinical development of several inhibitory compounds. Recently, high CALCRL expression has been shown to be associated with a poor prognosis in acute myeloid leukemia (AML). We investigate, therefore, the functional role of the CGRP-CALCRL axis in AML. To this end, in silico analyses, human AML cell lines, primary patient samples, and a C57BL/6-based mouse model of AML are used. We find that CALCRL is up-regulated at relapse of AML, in leukemic stem cells (LSCs) versus bulk leukemic cells, and in LSCs versus normal hematopoietic stem cells. CGRP protects receptor-positive AML cell lines and primary AML samples from apoptosis induced by cytostatic drugs used in AML therapy, and this effect is inhibited by specific antagonists. Furthermore, the CGRP antagonist olcegepant increases differentiation and reduces the leukemic burden as well as key stem cell properties in a mouse model of AML. These data provide a basis for further investigations into a possible role of CGRP-CALCRL inhibition in the therapy of AML.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/metabolismo , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteína Similar al Receptor de Calcitonina/antagonistas & inhibidores , Línea Celular Tumoral , Daunorrubicina/farmacología , Daunorrubicina/uso terapéutico , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Piperazinas , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Transducción de SeñalRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous disease with respect to its genetic and molecular basis and to patients´ outcome. Clinical, cytogenetic, and mutational data are used to classify patients into risk groups with different survival, however, within-group heterogeneity is still an issue. Here, we used a robust likelihood-based survival modeling approach and publicly available gene expression data to identify a minimal number of genes whose combined expression values were prognostic of overall survival. The resulting gene expression signature (4-GES) consisted of 4 genes (SOCS2, IL2RA, NPDC1, PHGDH), predicted patient survival as an independent prognostic parameter in several cohorts of AML patients (total, 1272 patients), and further refined prognostication based on the European Leukemia Net classification. An oncogenic role of the top scoring gene in this signature, SOCS2, was investigated using MLL-AF9 and Flt3-ITD/NPM1c driven mouse models of AML. SOCS2 promoted leukemogenesis as well as the abundance, quiescence, and activity of AML stem cells. Overall, the 4-GES represents a highly discriminating prognostic parameter in AML, whose clinical applicability is greatly enhanced by its small number of genes. The newly established role of SOCS2 in leukemia aggressiveness and stemness raises the possibility that the signature might even be exploitable therapeutically.
Asunto(s)
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Supresoras de la Señalización de Citocinas/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Ratones , Células Madre Neoplásicas/patología , PronósticoRESUMEN
BACKGROUND: The majority of individuals with acute myeloid leukemia (AML) respond to initial chemotherapy and achieve a complete remission, yet only a minority experience long-term survival because a large proportion of patients eventually relapse with therapy-resistant disease. Relapse therefore represents a central problem in the treatment of AML. Despite this, and in contrast to the extensive knowledge about the molecular events underlying the process of leukemogenesis, information about the mechanisms leading to therapy resistance and relapse is still limited. PURPOSE AND CONTENT OF REVIEW: Recently, a number of studies have aimed to fill this gap and provided valuable information about the clonal composition and evolution of leukemic cell populations during the course of disease, and about genetic, epigenetic, and gene expression changes associated with relapse. In this review, these studies are summarized and discussed, and the data reported in them are compiled in order to provide a resource for the identification of molecular aberrations recurrently acquired at, and thus potentially contributing to, disease recurrence and the associated therapy resistance. This survey indeed uncovered genetic aberrations with known associations with therapy resistance that were newly gained at relapse in a subset of patients. Furthermore, the expression of a number of protein coding and microRNA genes was reported to change between diagnosis and relapse in a statistically significant manner. CONCLUSIONS: Together, these findings foster the expectation that future studies on larger and more homogeneous patient cohorts will uncover pathways that are robustly associated with relapse, thus representing potential targets for rationally designed therapies that may improve the treatment of patients with relapsed AML, or even facilitate the prevention of relapse in the first place.
Asunto(s)
Leucemia Mieloide Aguda/patología , Células Clonales , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Humanos , Leucemia Mieloide Aguda/genética , RecurrenciaRESUMEN
RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3'-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a-binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a-binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. Cancer Res; 76(12); 3644-54. ©2016 AACR.
Asunto(s)
Leucemia Mieloide Aguda/patología , MicroARNs/fisiología , Proteínas de Unión a Fosfatidiletanolamina/fisiología , Línea Celular Tumoral , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Unión a Fosfatidiletanolamina/análisis , Proteínas de Unión a Fosfatidiletanolamina/genéticaRESUMEN
BACKGROUND: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. METHODS: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. RESULTS: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. CONCLUSIONS: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Leucemia Mieloide/patología , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunohistoquímica , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaAsunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide/genética , Células Madre Neoplásicas/metabolismo , Transcriptoma , Enfermedad Aguda , Adulto , Anciano , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , RecurrenciaRESUMEN
Even though a large proportion of patients with acute myeloid leukemia (AML) achieve a complete remission upon initial therapy, the majority of them eventually relapse with resistant disease. Overexpression of the gene coding for the transcription factor Ecotropic Virus Integration site 1 (EVI1) is associated with rapid disease recurrence and shortened survival. We therefore sought to identify EVI1 target genes that may play a role in chemotherapy resistance using a previously established in vitro model system for EVI1 positive myeloid malignancies. Gene expression microarray analyses uncovered the Cell Adhesion Molecule 1 (CADM1) gene as a candidate whose deregulation by EVI1 may contribute to drug refractoriness. CADM1 is an apoptosis inducing tumor suppressor gene that is inactivated by methylation in a variety of tumor types. In the present study we provide evidence that it may play a role in chemotherapy induced cell death in AML: CADM1 was induced by drugs used in the treatment of AML in a human myeloid cell line and in primary diagnostic AML samples, and its experimental expression in a cell line model increased the proportion of apoptotic cells. CADM1 up-regulation was abolished by ectopic expression of EVI1, and EVI1 expression correlated with increased CADM1 promoter methylation both in a cell line model and in primary AML cells. Finally, CADM1 induction was repressed in primary samples from AML patients at relapse. In summary, these data suggest that failure to up-regulate CADM1 in response to chemotherapeutic drugs may contribute to therapy resistance in AML.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/genética , Moléculas de Adhesión Celular/genética , Resistencia a Antineoplásicos/genética , Genes Supresores de Tumor/fisiología , Inmunoglobulinas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Anciano , Apoptosis/efectos de los fármacos , Molécula 1 de Adhesión Celular , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Persona de Mediana Edad , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proto-Oncogenes/genética , Factores de Transcripción/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The product of the ecotropic virus integration site 1 (EVI1) gene, whose overexpression is associated with a poor prognosis in myeloid leukemias and some epithelial tumors, regulates gene transcription both through direct DNA binding and through modulation of the activity of other sequence specific transcription factors. Previous results from our laboratory have shown that EVI1 influenced transcription regulation in response to the myeloid differentiation inducing agent, all-trans retinoic acid (ATRA), in a dual manner: it enhanced ATRA induced transcription of the RARß gene, but repressed the ATRA induction of the EVI1 gene itself. In the present study, we asked whether EVI1 would modulate the ATRA regulation of a larger number of genes, as well as biological responses to this agent, in human myeloid cells. U937 and HL-60 cells ectopically expressing EVI1 through retroviral transduction were subjected to microarray based gene expression analysis, and to assays measuring cellular proliferation, differentiation, and apoptosis. These experiments showed that EVI1 modulated the ATRA response of several dozens of genes, and in fact reinforced it in the vast majority of cases. A particularly strong synergy between EVI1 and ATRA was observed for GDF15, which codes for a member of the TGF-ß superfamily of cytokines. In line with the gene expression results, EVI1 enhanced cell cycle arrest, differentiation, and apoptosis in response to ATRA, and knockdown of GDF15 counteracted some of these effects. The potential clinical implications of these findings are discussed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Mieloides/metabolismo , Oncogenes , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Células HL-60 , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Células Mieloides/efectos de los fármacos , Proto-Oncogenes/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.
Asunto(s)
Leucemia Mieloide Aguda/genética , MicroARNs/biosíntesis , Adolescente , Adulto , Anciano , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , MicroARNs/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Adulto JovenRESUMEN
Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.