Emilios Tahinci

Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α

Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

In vertebrates, PDGFA and its receptor, PDGFRα, are expressed in the early embryo. Impairing their function causes an array of developmental defects, but the underlying target processes that are directly controlled by these factors are not well known. We show that in the Xenopus gastrula, PDGFA/PDGFRα signaling is required for the directional migration of mesodermal cells on the extracellular matrix of the blastocoel roof. Blocking PDGFRα function in the mesoderm does not inhibit migration per se, but results in movement that is randomized and no longer directed towards the animal pole. Likewise, compromising PDGFA function in the blastocoel roof substratum abolishes directionality of movement. Overexpression of wild-type PDGFA, or inhibition of PDGFA both lead to randomized migration, disorientation of polarized mesodermal cells, decreased movement towards the animal pole, and reduced head formation and axis elongation. This is consistent with an instructive role for PDGFA in the guidance of mesoderm migration.

Lrp6 is required for convergent extension during Xenopus gastrulation

Wnt signaling regulates β-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the β-catenin arm. We show that Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. We show that Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells and that Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, we identify the region of the Lrp6 protein with Wnt/PCP activity to a stretch of 36 amino acids, distinct from regions required for Wnt/β-catenin signaling. We propose a model in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/β-catenin signaling.

Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity.

A Xenopus reconstitution system reveals that the G protein Gβγ subunit contributes to β-catenin stabilization. Gβγ for β-Catenin Stability G proteins influence the Wnt–β-catenin pathway, which regulates various developmental processes; aberrant activity is associated with some cancers. The ligand Wnt interacts with a receptor complex that includes the seven-transmembrane protein Frizzled and the single-transmembrane protein LRP6 to activate the transcriptional regulatory activity of β-catenin. By screening the activity of purified G protein subunits in a Xenopus egg extract system, Jernigan et al. found that, in addition to a subset of Gα subunits, the Gβγ subunit also stabilized β-catenin. Various biochemical analyses, including analysis of transfected mammalian cells and in vitro assays, along with the use of a Gβγ-selective inhibitor, suggested that Gβγ recruited the kinase GSK3 to the membrane. After membrane recruitment, GSK3 phosphorylated LRP6, which then inhibited the β-catenin degradation complex, allowing β-catenin to translocate to the nucleus and activate transcription. Additionally, the Gβγ inhibitor prevented axis duplication of Xenopus embryos under conditions of excess LRP6 activity, thus verifying in vivo a role for Gβγ in this pathway. The Gβγ inhibitor failed to block Wnt-mediated activation of β-catenin, which suggests that a receptor other than Frizzled may activate the G protein that contributes to β-catenin signaling. Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate–binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator β-catenin. We screened the α and βγ subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes β-catenin degradation. We found that Gαo, Gαq, Gαi2, and Gβγ inhibited β-catenin degradation. Gβ1γ2 promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor–related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-βARK (C-terminal domain of β-adrenergic receptor kinase), an inhibitor of Gβγ, blocked LRP6 activity. Several components of the Wnt–β-catenin pathway formed a complex: Gβ1γ2, LRP6, GSK3, axin, and dishevelled. We propose that free Gβγ and Gα subunits, released from activated G proteins, act cooperatively to inhibit β-catenin degradation and activate β-catenin–mediated transcription.

Myeloid translocation gene family members associate with T-cell factors (TCFs) and influence TCF-dependent transcription.

ABSTRACT Canonical Wnt signaling is mediated by a molecular “switch” that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of β-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of β-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of β-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time‐lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases. Developmental Dynamics 235:1090–1099, 2006.

A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

ΔNp63 antagonizes p53 to regulate mesoderm induction in Xenopus laevis

p63, a homolog of the tumor suppressor p53, is critical for the development and maintenance of complex epithelia. The developmentally regulated p63 isoform, DeltaNp63, can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological roles is unclear. Based on our initial finding that the mesoderm-inducing factor activin A is suppressed by DeltaNp63 in human keratinocytes, we investigated the role of DeltaNp63 in regulating mesoderm induction during early Xenopus laevis development. We find that down-regulation of DeltaNp63 by morpholino injection in the early Xenopus embryo potentiates mesoderm formation whereas ectopic expression of DeltaNp63 inhibits mesoderm formation. Furthermore, we show that mesodermal induction after down-regulation of DeltaNp63 is dependent on p53. We propose that a key function for p63 in defining a squamous epithelial phenotype is to actively suppress mesodermal cell fates during early development. Collectively, we show that there is a distinct requirement for different p53 family members during the development of both mesodermal and ectodermal tissues. These findings have implications for the role of p63 and p53 in both development and tumorigenesis of human epithelia.