RESUMEN
High energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown. Here we show that the E3 ligase CRL5 Ozz , a member of the UPS, and its substrate Alix control the mitochondrial concentration of Slc25A4, a solute carrier that is essential for ATP production. The mitochondria in Ozz -/- or Alix -/- skeletal muscle share overt morphologic alterations (they are supernumerary, swollen, and dysmorphic) and have abnormal metabolomic profiles. We found that CRL5 Ozz ubiquitinates Slc25A4 and promotes its proteasomal degradation, while Alix facilitates SLC25A4 loading into exosomes destined for lysosomal destruction. The loss of Ozz or Alix offsets steady-state levels of Slc25A4, which disturbs mitochondrial metabolism and alters muscle fiber composition. These findings reveal hitherto unknown regulatory functions of Ozz and Alix in mitochondrial proteostasis.
RESUMEN
Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.
Asunto(s)
Neuraminidasa , Rabdomiosarcoma , Animales , Haploinsuficiencia , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas , Lisosomas/metabolismo , Ratones , Neuraminidasa/genética , Neuraminidasa/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Microambiente TumoralRESUMEN
Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.
Asunto(s)
Rabdomiosarcoma Alveolar , Rabdomiosarcoma , Carcinogénesis/genética , Línea Celular Tumoral , Niño , Proteína Forkhead Box O1/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción Paired Box/uso terapéutico , Rabdomiosarcoma/genética , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patologíaRESUMEN
During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.
RESUMEN
Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.
Asunto(s)
Autofagia/fisiología , Epigénesis Genética , Lisosomas/metabolismo , Biogénesis de Organelos , Politetrafluoroetileno/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Sitios de Unión , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasa 2/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Células Madre , Transcripción GenéticaRESUMEN
Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development. Therefore, we investigated whether the protein synthesis machinery that is regulated by mTORC1 (mTOR complex 1) signaling can in turn regulate mTORC1 activity. We found that inhibition of protein synthesis results in rapid activation of mTORC1 signaling, thereby uncovering a feedback loop between mTOR and the translation machinery. This mTORC1 activation requires tuberous sclerosis complex (TSC) but is independent of AKT. In addition, by using a PKC-δ (protein kinase c delta)-specific inhibitor and PKC-δ siRNA knockdown, we found that PKC-δ kinase activity is required for mTORC1 activation in response to translation inhibitors. Furthermore, translation inhibition activates PKC-δ. Subsequently, we investigated whether PKC-δ can phosphorylate and inactivate TSC1/2, leading to mTORC1 activation. In vitro kinase assays showed direct phosphorylation of TSC2 (S932 and S939) by PKC-δ, which was confirmed by mass spectrometry. In vivo kinase analysis further indicated that both S932 and S939 are phosphorylated in response to translation inhibitors. Finally, phosphorylation defective TSC2 mutants (S932A and S939A single mutants and a S932A/S939A double mutant) failed to upregulate mTORC1 activity in the presence of translation inhibitors, suggesting that activation of mTORC1 by translation inhibitors is mediated by PKC-δ phosphorylation of TSC2 at S932/S939, which inactivates TSC.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína Quinasa C-delta/metabolismo , Transducción de Señal , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Activación Enzimática , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mutación Missense , Fosforilación , Proteína Quinasa C-delta/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
The ETS transcription factor ETV7 has been characterized as a hematopoietic oncoprotein, which requires cooperating mutations for its leukemogenic activity. Although the ETV7 gene is highly conserved among vertebrates, part of the rodents, including Mus musculus, deleted the Etv7 gene locus. Many human hematopoietic malignancies upregulate ETV7 expression but contrary to ETV7's role in oncogenesis, its physiological role in normal tissues is unknown. To determine the physiological function of ETV7 in vivo and determine its role in tumorigenesis in a mouse model, we have generated an ETV7 transgenic mouse that carries a single copy of human BAC DNA containing the ETV7 gene locus and its regulatory sequences. ETV7 heterozygous (ETV7Tg+/WT) mice were fertile, normal in size and born at a normal Mendelian frequency. They had a normal blood count, did not display any gross physical or behavioral abnormalities, and were not tumor-prone. The ETV7 expression pattern in hematopoietic cells of ETV7Tg+/WT mice is very similar to that in human hematopoietic cells. To examine the oncogenic potential of ETV7 in vivo, we crossed ETV7Tg+/WT mice with tumor-prone mouse models. ETV7 greatly accelerated loss of Pten (phosphatase and tensin homolog)-evoked leukemogenesis in PtenΔ/ΔETV7Tg+/WT mice after deletion of the conditional Pten allele. Consistent with this observation, ETV7 expression enhanced the colony-forming and self-renewal activities of primary myeloid Pten-/- cells. In this study we established a transgenic mouse in which we can more accurately model ETV7-associated human tumorigenesis in vivo.
Asunto(s)
Carcinogénesis/genética , Ratones Transgénicos/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-ets/genética , Animales , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Heterocigoto , Humanos , Ratones , Células Mieloides/patología , Neoplasias/patología , Fosfohidrolasa PTEN/genéticaRESUMEN
The mechanistic target of rapamycin (mTOR) serine/threonine kinase, a critical regulator of cell proliferation, is frequently deregulated in human cancer. Although rapamycin inhibits the two canonical mTOR complexes, mTORC1 and mTORC2, it often shows minimal benefit as an anticancer drug. This is caused by rapamycin resistance of many different tumors, and we show that a third mTOR complex, mTORC3, contributes to this resistance. The ETS (E26 transformation-specific) transcription factor ETV7 interacts with mTOR in the cytoplasm and assembles mTORC3, which is independent of ETV7's transcriptional activity. This complex exhibits bimodal mTORC1/2 activity but is devoid of crucial mTORC1/2 components. Many human cancers activate mTORC3 at considerable frequency, and tumor cell lines that lose mTORC3 expression become rapamycin-sensitive. We show mTORC3's tumorigenicity in a rhabdomyosarcoma mouse model in which transgenic ETV7 expression accelerates tumor onset and promotes tumor penetrance. Discovery of mTORC3 represents an mTOR paradigm shift and identifies a novel target for anticancer drug development.
Asunto(s)
Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Proto-Oncogénicas c-ets/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.
Asunto(s)
Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/genética , Translocación Genética/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ratones , Mioblastos/metabolismo , Mioblastos/patología , ARN Mensajero/biosíntesis , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patologíaRESUMEN
ETV7 is a human oncoprotein that cooperates with Eµ-MYC to promote pre-B-cell leukemia in mice. It is normally expressed in the bone marrow and fetal liver and is upregulated in primary leukemia, suggesting that it is involved in proper hematopoiesis and leukemogenesis. ETV7 has been deleted in most rodents, but is conserved in all other vertebrates, including the zebrafish, Danio rerio. In this report, we characterize the function of the zebrafish etv7 gene during erythropoiesis. Our results demonstrate that etv7 regulates the expression of the zebrafish lanosterol synthase (lss) gene, an essential gene in the cholesterol synthesis pathway. Furthermore, morpholino knockdown of etv7 leads to loss of hemoglobin-containing red blood cells, a phenotype that can be rescued by injection of exogenous cholesterol. We conclude that etv7 is essential for normal red blood cell development through regulation of the lss gene and the cholesterol synthesis pathway.
Asunto(s)
Vías Biosintéticas , Colesterol/biosíntesis , Eritrocitos/metabolismo , Eritropoyesis , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Eritrocitos/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hemoglobinas/metabolismo , Humanos , Ratones , Morfolinos/farmacología , Proteínas Proto-Oncogénicas c-ets/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.
Asunto(s)
Proteína MioD/genética , Mioblastos Esqueléticos/patología , Células Madre Neoplásicas/patología , Factor de Transcripción PAX7/genética , Rabdomiosarcoma/patología , Animales , Regulación Neoplásica de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Desarrollo de Músculos/genética , Células Madre Neoplásicas/enzimología , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/genética , Rabdomiosarcoma/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.
Asunto(s)
Apoptosis/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Rabdomiosarcoma Alveolar/genética , Animales , Compuestos de Bifenilo/farmacología , Ácidos Borónicos/administración & dosificación , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Nitrofenoles/farmacología , Oligopéptidos/farmacología , Piperazinas/farmacología , Pirazinas/administración & dosificación , Pirazinas/farmacología , Rabdomiosarcoma Alveolar/mortalidad , Rabdomiosarcoma Alveolar/patología , Sulfonamidas/farmacología , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1's in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12-228 (Δ12-228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12-228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12-228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.
Asunto(s)
Secuencia de Aminoácidos , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Células Mieloides/metabolismo , Proteínas de Neoplasias/genética , Proteínas Oncogénicas/genética , Eliminación de Secuencia , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Recuento de Células , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Perfilación de la Expresión Génica , Vectores Genéticos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Datos de Secuencia Molecular , Células Mieloides/patología , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Oncogénicas/metabolismo , Retroviridae/genética , Análisis de Supervivencia , Transactivadores , Transducción Genética , Proteínas Supresoras de TumorRESUMEN
Rhabdomyosarcoma is a soft tissue sarcoma arising from cells of a mesenchymal or skeletal muscle lineage. Alveolar rhabdomyosarcoma (ARMS) is more aggressive than the more common embryonal (ERMS) subtype. ARMS is more prone to metastasis and carries a poorer prognosis. In contrast to ERMS, the majority of ARMS tumors carry one of several characteristic chromosomal translocations, such as t(2;13)(q35;q14), which results in the expression of a PAX3-FOXO1 fusion transcription factor. In this review we discuss the genes that cooperate with PAX3-FOXO1, as well as the target genes of the fusion transcription factor that contribute to various aspects of ARMS tumorigenesis. The characterization of these pathways will lead to a better understanding of ARMS tumorigenesis and will allow the design of novel targeted therapies that will lead to better treatment for this aggressive pediatric tumor.
RESUMEN
The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that â¼20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, â¼50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Animales , Recuperación de Fluorescencia tras Fotoblanqueo , Ratones , Método de Montecarlo , Células 3T3 NIH , Proteínas Oncogénicas/genética , Proteínas de Fusión Oncogénica/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Transactivadores , Proteínas Supresoras de Tumor , Proteína ETS de Variante de Translocación 6RESUMEN
We and others have identified FGFR4 as a direct transcriptional target of the alveolar rhabdomyosarcoma (ARMS) specific fusion protein, PAX3-FOXO1. We hypothesized fibroblast growth factor receptor 4 (FGFR4) may act as an effector of PAX3-FOXO1, contributing to PAX3-FOXO1 tumorigenic phenotypes. However, we demonstrate that enhanced expression of FGFR4 does not contribute to inhibited differentiation, enhanced proliferation, or transformation downstream of PAX3-FOXO1 in primary mouse myoblasts. Therefore we were unable to identify any contribution of up regulation of wild type FGFR4 to PAX3-FOXO1 driven tumorigenesis. Conversely, a constitutively active mutant of FGFR4 can enhance primary myoblast proliferation and transformation, indicating activating mutations of FGFR4 could contribute to the development and progression of ARMS. We sequenced the FGFR4 mRNA from five ARMS cell lines and identified no somatic mutations, nor any association with any human single nucleotide polymorphism within the FGFR4 coding region.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Rabdomiosarcoma Alveolar/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Mutación , Mioblastos/metabolismo , Mioblastos/patología , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Polimorfismo de Nucleótido Simple , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patología , Regulación hacia ArribaRESUMEN
Alveolar rhabdomyosarcoma (ARMS) is a muscle-derived childhood tumor characterized by production of oncogenic PAX3/7-FOXO1 chimeric transcription factors. While downstream targets of the PAX3-FOXO1 oncoprotein in ARMS have been defined, the functional relevance of these targets is unclear. Here, we show that upregulation of the cannabinoid receptor 1 (Cnr1/Cb1) by PAX3-FOXO1 in mouse primary myoblasts and ARMS cell lines, contributes to PAX3-FOXO1 phenotypes, both in vivo and in vitro. In primary myoblasts, Cnr1 was dispensable for PAX3-FOXO1 to mediate cell proliferation, differentiation, or transformation; however, Cnr1 function was essential to increase the invasive capacity conferred by PAX3-FOXO1 overexpression in these cells. Genetic or pharmacologic abrogation of Cnr1 inhibited the enhanced basement membrane invasion induced by PAX3-FOXO1. Cnr1 loss by either route also dramatically reduced lung metastasis formation. Taken together, our findings strongly implicate Cnr1 as a novel tractable target to inhibit ARMS invasion and metastasis.
Asunto(s)
Movimiento Celular , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children with an annual incidence of five new cases per million. Alveolar rhabdomyosarcoma (ARMS) is characterized by the t(2;13) or t(1;13) chromosomal translocations, which generate the PAX3-FOXO1 or PAX7-FOXO1 fusion genes, respectively. The oncogenic activity of PAX3-FOXO1 has been demonstrated in vitro and in vivo, yet expression of the fusion protein alone in primary myoblasts or a mouse model is insufficient for tumorigenic transformation. To identify genes cooperating with PAX3-FOXO1 in ARMS tumorigenesis, we generated a retroviral complementary DNA (cDNA) expression library from the Rh30 ARMS cell line. Arf-/- myoblasts expressing PAX3-FOXO1 and the retroviral cDNA library rapidly formed tumors after subcutaneous injection into NOD-SCID mice. Tumors formed by Arf-/-/PAX3-FOXO1/MarX-library myoblasts contained an unknown cDNA, encoding the C-terminus of the Homo sapiens hypothetical protein, FLJ10404, herein named IRIZIO. Expression of full length IRIZIO cDNA also cooperated with PAX3-FOXO1 in the transformation of Arf-/- myoblasts. Given that IRIZIO is expressed at increased levels in RMS, it might contribute to rhabdomyosarcomagenesis in humans.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Oncogenes , Factores de Transcripción Paired Box/fisiología , Rabdomiosarcoma Alveolar/etiología , Animales , Línea Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones SCID , Mioblastos/patología , Proteínas de Neoplasias/genética , ARN Mensajero/análisis , Proteína de Retinoblastoma/fisiología , Rabdomiosarcoma Alveolar/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Forced expression of MN1 in primitive mouse hematopoietic cells causes acute myeloid leukemia and impairs all-trans retinoic acid-induced granulocytic differentiation. Here, we studied the effects of MN1 on myeloid differentiation and proliferation using primary human CD34(+) hematopoietic cells, lineage-depleted mouse bone marrow cells, and bipotential (granulocytic/monocytic) human acute myeloid leukemia cell lines. We show that exogenous MN1 stimulated the growth of CD34(+) cells, which was accompanied by enhanced survival and increased cell cycle traverse in cultures supporting progenitor cell growth. Forced MN1 expression impaired both granulocytic and monocytic differentiation in vitro in primary hematopoietic cells and acute myeloid leukemia cell lines. Endogenous MN1 expression was higher in human CD34(+) cells compared with both primary and in vitro-differentiated monocytes and granulocytes. Microarray and real-time reverse-transcribed polymerase chain reaction analysis of MN1-overexpressing CD34(+) cells showed down-regulation of CEBPA and its downstream target genes. Reintroduction of conditional and constitutive CEBPA overcame the effects of MN1 on myeloid differentiation and inhibited MN1-induced proliferation in vitro. These results indicate that down-regulation of CEBPA activity contributes to MN1-modulated proliferation and impaired myeloid differentiation of hematopoietic cells.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular/genética , Proliferación Celular , Células Madre Hematopoyéticas/fisiología , Células Mieloides/fisiología , Proteínas Supresoras de Tumor/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colecalciferol/farmacología , Células HL-60 , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Transactivadores , Transfección , Tretinoina/farmacología , Proteínas Supresoras de Tumor/metabolismo , Células U937RESUMEN
High levels of expression of the human DEK gene have been correlated with numerous human malignancies. Intracellular DEK functions have been described in vitro and include DNA supercoiling, DNA replication, RNA splicing, and transcription. We have shown that DEK also suppresses cellular senescence, apoptosis, and differentiation, thus promoting cell growth and survival in monolayer and organotypic epithelial raft models. Such functions are likely to contribute to cancer, but direct evidence to implicate DEK as an oncogene has remained elusive. Here, we show that in line with an early role in tumorigenesis, murine papilloma formation in a classical chemical carcinogenesis model was reduced in DEK knockout mice. Additionally, human papillomavirus E6/E7, hRas, and DEK cooperated in the transformation of keratinocytes in soft agar and xenograft establishment, thus also implicating DEK in tumor promotion at later stages. Finally, adenoviral DEK depletion via short hairpin RNA expression resulted in cell death in human tumor cells in vitro and in vivo, but did not significantly affect differentiated epithelial cells. Taken together, our data uncover oncogenic DEK activities as postulated from its frequent up-regulation in human malignancies, and suggest that the targeted suppression of DEK may become a strategic approach to the treatment of cancer.