Hunk negatively regulates c-myc to promote Akt-mediated cell survival and mammary tumorigenesis induced by loss of Pten.

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.

Hes6 is required for MyoD induction during gastrulation.

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.

Tumor metastasis: a new twist on epithelial-mesenchymal transitions.

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.

The developmental expression of cell cycle regulators in Xenopus laevis.

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.

Deciphering the molecular basis of breast cancer metastasis with mouse models.

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.

Slug stability is dynamically regulated during neural crest development by the F-box protein Ppa.

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.

Notch targets the Cdk inhibitor Xic1 to regulate differentiation but not the cell cycle in neurons.

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.

Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells.

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.

A single cdk inhibitor, p27Xic1, functions beyond cell cycle regulation to promote muscle differentiation in Xenopus.

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.

The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus.

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.

G1/S phase cyclin-dependent kinase overexpression perturbs early development and delays tissue-specific differentiation in Xenopus.

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.

Hes6 regulates myogenic differentiation.

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.