Hiroki Hikasa

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G1 and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

Frodo interacts with Dishevelled to transduce Wnt signals

Dishevelled (Dsh) is required for the specification of cell fate and polarity by secreted Wnt proteins. Frodo, a novel conserved Dsh-binding protein, synergized with Xenopus Dsh (XDsh) in secondary axis induction in Xenopus laevis embryos. A dominant inhibitory construct and antisense oligonucleotide-mediated depletion of Frodo inhibited axial development in response to XDsh and XWnt8, and suppressed transcriptional activation of a reporter construct. At later embryonic stages, both dominant negative Frodo and antisense oligonucleotides interfered with the expression of regional neural markers and caused eye deficiencies, indicating that Frodo is required for normal eye and neural tissue development. Full-length Frodo RNA suppressed these loss-of-function phenotypes, attesting to their specificity. These findings establish a function for Frodo as an essential positive regulator of Wnt signalling.

Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.

Phosphorylation of TCF Proteins by Homeodomain-interacting Protein Kinase 2

Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos.

Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a(Δ/Δ)1b(tr/+) or Mob1a(Δ/+)1b(tr/tr) mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.

The involvement of Frodo in TCF-dependent signaling and neural tissue development

Frodo is a novel conserved regulator of Wnt signaling that has been identified by its association with Dishevelled, an intracellular component of Wnt signal transduction. To understand further how Frodo functions, we have analyzed its role in neural development using specific morpholino antisense oligonucleotides. We show that Frodo and the closely related Dapper synergistically regulate head development and morphogenesis. Both genes were cell-autonomously required for neural tissue formation, as defined by the pan-neural markers sox2 and nrp1. By contrast,β -catenin was not required for pan-neural marker expression, but was involved in the control of the anteroposterior patterning. In the mesoderm, Frodo and Dapper were essential for the expression of the organizer genes chordin, cerberus and Xnr3, but they were not necessary for the expression of siamois and goosecoid, established targets of β-catenin signaling. Embryos depleted of either gene showed a decreased transcriptional response to TCF3-VP16, aβ -catenin-independent transcriptional activator. Whereas the C terminus of Frodo binds Dishevelled, we demonstrate that the conserved N-terminal domain associates with TCF3. Based on these observations, we propose that Frodo and Dapper link Dsh and TCF to regulate Wnt target genes in a pathway parallel to that of β-catenin.

Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice

Significance Patients with intrahepatic cholangiocellular carcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (cHC-CC) have worse prognoses than those with hepatocellular carcinoma and rarely show clinical responses to drugs. Our analyses of mice with liver-specific deletions of Mps One Binder Kinase Activator (MOB)1A/1B reveal that MOB1A/1B constitute the most important hub of Hippo signaling in mammalian liver. MOB1A/1B maintain hepatocyte stem/progenitor cell quiescence and are potent tumor suppressors, especially in cHC-CCs and ICCs. Because these functions depend on the Hippo target Yap1/Taz and the Yap1/Taz targets Tgfbs, our data point to a new therapeutic approach for liver cancer based on inhibition of MOB1-YAP1/TAZ and/or TGF-βs–SMADs signaling. Our demonstration that well-tolerated and already-approved antiparasitic drugs inhibit YAP1 signaling may point to a new route of treatment for these cancers that can be rapidly tested and implemented. Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial–mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs.

Two Frodo/Dapper homologs are expressed in the developing brain and mesoderm of zebrafish

Members of the Wnt family of extracellular proteins play essential roles during many phases of vertebrate embryonic development. The molecular mechanism of their action involves a complex cascade of intracellular signaling events, which remains to be understood completely. Recently, two novel cytoplasmic modulators of Wnt signaling, Frodo and Dapper, were identified in Xenopus. We report isolation of their homologs in zebrafish, and show that these genes, frd1 and frd2, are expressed in restricted domains during embryogenesis. Both genes are expressed during early gastrulation in the future mesendoderm, and continue to be expressed in distinct patterns in the forming neurectoderm and mesoderm. Comparative sequence analysis and similar expression patterns argue that frd1 is the zebrafish ortholog of Frodo and Dapper, whereas frd2 is a more divergent member of the same family. Our data suggest important roles for zebrafish frd1 and frd2 in patterning the neural plate and several mesodermal derivatives. Developmental Dynamics 230:403–409, 2004.

Vertebrate homologues of Frodo are dynamically expressed during embryonic development in tissues undergoing extensive morphogenetic movements.

Frodo has been identified as a protein interacting with Dishevelled, an essential mediator of the Wnt signaling pathway, critical for the determination of cell fate and polarity in embryonic development. In this study, we use specific gene probes to characterize stage‐ and tissue‐specific expression patterns of the mouse Frodo homologue and compare them with Frodo expression patterns in Xenopus embryos. In situ hybridization analysis of mouse Frodo transcripts demonstrates that, similar to Xenopus Frodo, mouse Frodo is expressed in primitive streak mesoderm, neuroectoderm, neural crest, presomitic mesoderm, and somites. In many cases, Frodo expression is confined to tissues undergoing extensive morphogenesis, suggesting that Frodo may be involved in the regulation of cell shape and motility. Highly conserved dynamic expression patterns of Frodo homologues indicate a similar function for these proteins in different vertebrates. Developmental Dynamics 235:279–284, 2006.

Reorganization of actin cytoskeleton by FRIED, a Frizzled-8 associated protein tyrosine phosphatase.

Frizzled receptors transduce signals from the extracellular Wnt ligands through multiple signaling pathways that affect cytoskeletal organization and regulate gene expression. Direct intracellular mediators of Frizzled signaling are largely unknown. We identified FRIED (Frizzled interaction and ectoderm defects) by its association with the C‐terminal PDZ‐binding motif of Xenopus Frizzled 8. FRIED contains an N‐terminal KIND domain, a FERM domain, six PDZ domains, and a tyrosine phosphatase domain, being similar in structure to the protein tyrosine phosphatase PTP‐BAS/PTP‐BL. We report that FRIED proteins with the FERM domain localize to the apical cortex and can inhibit Wnt8‐mediated, but not β‐catenin‐mediated, secondary axis induction in Xenopus embryos, suggesting a specific interaction with Wnt signaling. A FRIED construct containing the FERM domain induced reorganization of pigment granules and cortical actin in Xenopus ectoderm. Wnt5a suppressed the depigmentation of ectoderm triggered by FRIED, demonstrating that Wnt5a and FRIED functionally interact to regulate the cytoskeletal organization. Our data are consistent with the possibility that FRIED functions by modulating Rac1 activity. We propose that FRIED is an adaptor protein that serves as a molecular link between Wnt signaling and actin cytoskeleton. Developmental Dynamics 234:90–101, 2005.