RESUMEN
The protooncogenes Akt and c-myc each positively regulate cell growth and proliferation, but have opposing effects on cell survival. These oncogenes cooperate to promote tumorigenesis, in part because the prosurvival effects of Akt offset the proapoptotic effects of c-myc. Akt's ability to counterbalance c-myc's proapoptotic effects has primarily been attributed to Akt-induced stimulation of prosurvival pathways that indirectly antagonize the effects of c-myc. We report a more direct mechanism by which Akt modulates the proapoptotic effects of c-myc. Specifically, we demonstrate that Akt up-regulates the adenosine monophosphate-associated kinase (AMPK)-related protein kinase, Hormonally up-regulated neu-associated kinase (Hunk), which serves as an effector of Akt prosurvival signaling by suppressing c-myc expression in a kinase-dependent manner to levels that are compatible with cell survival. Consequently, Akt pathway activation in the mammary glands of Hunk(-/-) mice results in induction of c-myc expression to levels that induce apoptosis. c-myc knockdown rescues the increase in apoptosis induced by Hunk deletion in cells in which Akt has been activated, indicating that repression of c-myc is a principal mechanism by which Hunk mediates the prosurvival effects of Akt. Consistent with this mechanism of action, we find that Hunk is required for c-myc suppression and mammary tumorigenesis induced by phosphatase and tensin homolog (Pten) deletion in mice. Together, our findings establish a prosurvival function for Hunk in tumorigenesis, define an essential mechanism by which Akt suppresses c-myc-induced apoptosis, and identify Hunk as a previously unrecognized link between the Akt and c-myc oncogenic pathways.
Asunto(s)
Neoplasias Mamarias Animales/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Apoptosis , Supervivencia Celular , Eliminación de Gen , Neoplasias Mamarias Animales/genética , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Transcripción Genética , Regulación hacia ArribaRESUMEN
The specification of mesoderm into distinct compartments sharing the same lineage restricted fates is a crucial step occurring during gastrulation, and is regulated by morphogenic signals such as the FGF/MAPK and activin pathways. One target of these pathways is the transcription factor XmyoD, which in early gastrulation is expressed in the lateral and ventral mesoderm. Expression of the hairy/enhancer of split transcription factor hes6, is also restricted to lateral and ventral mesoderm in gastrula stage Xenopus embryos, leading us to investigate whether it has a role in XmyoD regulation. In vivo, Xhes6 is required for FGF-mediated induction of XmyoD expression but not for induction of early mesoderm. The WRPW domain of Xhes6, which binds Groucho family transcriptional co-regulators, is essential for the XmyoD-inducing activity of Xhes6. Two Groucho proteins, Xgrg2 and Xgrg4, are expressed in lateral and ventral mesoderm, and inhibit expression of XmyoD. Xhes6 binds both Xgrg2 and Xgrg4 and relieves their inhibition of XmyoD expression. We also find that lowering Xhes6 expression levels blocks normal myogenic differentiation at tail bud stage. We conclude that Xhes6 is essential for XmyoD induction and acts by relieving Groucho-mediated repression of gene expression.
Asunto(s)
Gastrulación , Regulación del Desarrollo de la Expresión Génica , Proteína MioD/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Activinas/metabolismo , Animales , Tipificación del Cuerpo/efectos de los fármacos , Proteínas Co-Represoras , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/enzimología , Factores de Crecimiento de Fibroblastos/farmacología , Gastrulación/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Proteína Nodal , Oligonucleótidos Antisentido/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMEN
Epithelial-mesenchymal transitions are essential for normal embryonic development and for progression of non-invasive tumor cells into malignant, metastatic carcinomas. Twist, an important regulator of morphogenesis in the embryo, has recently been implicated in the onset of invasive behavior during tumor progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Modelos Biológicos , Metástasis de la Neoplasia/fisiopatología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Epitelio/fisiología , Mesodermo/fisiología , Ratones , Proteínas Nucleares/fisiología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/fisiología , Proteína 1 Relacionada con TwistRESUMEN
Several cyclins and cdks have been cloned in Xenopus, but their developmental expression has not been thoroughly examined. We have analyzed the temporal and spatial expression of cdk1, cdk2, cdk4 and cyclins D1, D2, E, A1, A2 and B1 by in situ hybridization. The transcripts of these cyclins and cdks exhibit striking tissue-restricted expression patterns very early in development that cannot be strictly correlated with proliferation. While the cdks and their activating cyclins are expressed in somewhat overlapping patterns, they are not precisely coincident. Additionally, maternal and zygotic cyclin forms demonstrate markedly different expression patterns.
Asunto(s)
Proteínas de Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas , Animales , Proteína Quinasa CDC2/biosíntesis , Proteína Quinasa CDC2/genética , Quinasas CDC2-CDC28/biosíntesis , Quinasas CDC2-CDC28/genética , Proteínas de Ciclo Celular/biosíntesis , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , Ciclinas/biosíntesis , Ciclinas/genética , Perfilación de la Expresión Génica , Hibridación in Situ , Proteínas de Xenopus , Xenopus laevisRESUMEN
Breast cancer begins as a localized disease, but has the potential to spread to distant sites within the body. This process--known as metastasis--is the leading cause of death from breast cancer. Whether the ability of cancer cells to metastasize is an intrinsic or acquired feature is currently a topic of considerable debate. Nevertheless, the key cellular events required for metastasis are generally accepted. These include invasion of the surrounding stromal tissue, intravasation, evasion of programmed cell death, arrest within the vasculature at a distant site, extravasation, and establishment and growth within a new microenvironment. The development of mouse models that faithfully mimic critical aspects of human neoplasia has been instrumental in framing our current understanding of multistage carcinogenesis. This review examines the advantages and limitations of existing murine models for mammary carcinogenesis for probing the molecular mechanisms that contribute to metastasis, as well as non-invasive tumor imaging approaches to facilitate these investigations.
Asunto(s)
Neoplasias de la Mama/genética , Metástasis de la Neoplasia/genética , Animales , Neoplasias de la Mama/patología , Adhesión Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patologíaRESUMEN
The neural crest is a population of stem-cell-like precursors found only in vertebrates. Slug, a member of the Snail family of zincfinger transcriptional repressors, is a critical regulator of neural crest development and has also been implicated in the acquisition of invasive behavior during tumor progression. Despite its central role in these two important processes, little is known about the mechanisms that control the expression and/or activity of Slug. We demonstrate that Slug is a labile protein whose stability is positively reinforced through activation of the neural crest regulatory program. We identify Partner of paired (Ppa) as the F-box component of a modular E3 ligase, and show that it is expressed in neural crest-forming regions, and that it binds to and promotes ubiquitin-mediated proteasomal degradation of Slug. Misexpression of Ppa inhibits the formation of neural crest precursors, and Slug mutants in which Ppa binding has been abrogated rescue this inhibition. These results provide novel insight into the regulation of Slug, a protein that plays a central role in neural crest precursor formation, as well as in developmental and pathological epithelial to mesenchymal transitions.
Asunto(s)
Proteínas F-Box/metabolismo , Cresta Neural/embriología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Xenopus laevis/embriología , Dedos de Zinc , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas F-Box/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Cresta Neural/citología , Cresta Neural/metabolismo , Proteínas Represoras/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Ubiquitina/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The proneural protein neurogenin (XNGNR1) drives differentiation of primary neurons in combination with the cyclin-dependent kinase (Cdk) inhibitor Xic1. Differentiation is inhibited by Notch signalling, resulting in a scattered neuronal distribution. Here we show that Notch signalling regulates the level of Xic1 transcription, yet this does not correlate with Notch's ability to perturb the cell cycle. Instead, Notch may regulate Xic1 levels to control its differentiation function directly, which is required in parallel with XNGNR1 to promote primary neurogenesis. Indeed, Notch-mediated repression of both XNGNR1 and Xic1 must be relieved for neuronal differentiation to occur. Interestingly, although Xic1 is required for XNGNR1-mediated neurogenesis, it is not required for XNGNR1-mediated upregulation of Delta, allowing establishment of the negative feedback loop involved in lateral inhibition. Therefore, Notch targets Cdk inhibitor expression to regulate differentiation of primary neurons, and its effects on the cell cycle may be of secondary importance.
Asunto(s)
Ciclo Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Neuronas/citología , Receptores Notch/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Diferenciación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Neuronas/fisiología , Receptores Notch/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , beta-Galactosidasa/metabolismoRESUMEN
Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. Here, we show that the small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice/genética , Cresta Neural/embriología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Animales , Western Blotting , Genes myc/genética , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Diferenciación , Cresta Neural/citología , Oligonucleótidos , Células Madre/citología , Factores de Transcripción/genética , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
The molecular basis of the antagonism between cellular proliferation and differentiation is poorly understood. We have investigated the role of the cyclin-dependent kinase inhibitor p27(Xic1) in the co-ordination of cell cycle exit and differentiation during early myogenesis in vivo using Xenopus embryos. In this report, we demonstrate that p27(Xic1) is highly expressed in the developing myotome, that ablation of p27(Xic1) protein prevents muscle differentiation and that p27(Xic1) synergizes with the transcription factor MyoD to promote muscle differentiation. Furthermore, the ability of p27(Xic1) to promote myogenesis resides in an N-terminal domain and is separable from its cell cycle regulation function. This data demonstrates that a single cyclin-dependent kinase inhibitor, p27(Xic1), controls in vivo muscle differentiation in Xenopus and that regulation of this process by p27(Xic1) requires activities beyond cell cycle inhibition.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Músculos/embriología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Músculos/citología , Músculos/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
We have investigated the role of the cyclin-dependent kinase inhibitor, p27(Xic1), in the coordination of cell cycle exit and differentiation during early neurogenesis. We demonstrate that p27(Xic1) is highly expressed in cells destined to become primary neurones and is essential for an early stage of neurogenesis. Ablation of p27(Xic1) protein prevents differentiation of primary neurones, while overexpressing p27(Xic1) promotes their formation. p27(Xic1) may enhance neurogenesis by stabilising the bHLH protein, neurogenin. Moreover, the ability of p27(Xic1) to stabilise neurogenin and enhance neurogenesis localises to an N-terminal domain of the molecule and is separable from its ability to inhibit the cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inducción Embrionaria/fisiología , Neuronas/citología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
Cell division and differentiation are largely incompatible but the molecular links between the two processes are poorly understood. Here, we overexpress G1/S phase cyclins and cyclin-dependent kinases in Xenopus embryos to determine their effect on early development and differentiation. Overexpression of cyclin E prior to the midblastula transition (MBT), with or without cdk2, results in a loss of nuclear DNA and subsequent apoptosis at early gastrula stages. By contrast, overexpressed cyclin A2 protein does not affect early development and, when stabilised by binding to cdk2, persists to tailbud stages. Overexpression of cyclin A2/cdk2 in post-MBT embryos results in increased proliferation specifically in the epidermis with concomitant disruption of skin architecture and delay in differentiation. Moreover, ectopic cyclin A2/cdk2 also inhibits differentiation of primary neurons but does not affect muscle. Thus, overexpression of a single G1/S phase cyclin/cdk pair disrupts the balance between division and differentiation in the early vertebrate embryo in a tissue-specific manner.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Fase G1/fisiología , Fase S/fisiología , Xenopus laevis/embriología , Animales , Apoptosis/fisiología , Blástula/citología , Quinasas CDC2-CDC28/genética , Quinasas CDC2-CDC28/metabolismo , Diferenciación Celular/fisiología , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Embrión no Mamífero , Células Epidérmicas , Epidermis/embriología , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/citología , Músculo Esquelético/embriología , Neuronas/citología , Especificidad de Órganos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMEN
Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21(Cip1), and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.