N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine inhibits bladder cancer progression by suppressing YAP1/TAZ.

Bladder cancer (BlC) is the fourth most common cancer in males worldwide, but few systemic chemotherapy options for its effective treatment exist. The development of new molecularly-targeted agents against BlC is therefore an urgent issue. The Hippo signaling pathway, with its upstream LATS kinases and downstream transcriptional co-activators YAP1 and TAZ, plays a pivotal role in diverse cell functions, including cell proliferation. Recent studies have shown that overexpression of YAP1 occurs in advanced BlCs and is associated with poor patient prognosis. Accessing data from our previous screening of a chemical library of compounds targeting the Hippo pathway, we identified DMPCA (N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine) as an agent able to induce the phosphorylation of LATS1 and YAP1/TAZ in BlC cells, thereby suppressing their viability both in vitro and in mouse xenografts. Our data indicate that DMPCA has a potent anti-tumor effect, and raise the possibility that this agent may represent a new and effective therapeutic option for BlC.

Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation.

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

YAP1 is a potent driver of the onset and progression of oral squamous cell carcinoma.

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common group of cancers in the world, and patients have a poor prognosis. Here, we present data indicating that YAP1 may be a strong driver of the onset and progression of oral SCC (OSCC), a major subtype of HNSCC. Mice with tongue-specific deletion of Mob1a/b and thus endogenous YAP1 hyperactivation underwent surprisingly rapid and highly reproducible tumorigenesis, developing tongue carcinoma in situ within 2 weeks and invasive SCC within 4 weeks. In humans, precancerous tongue dysplasia displays YAP1 activation correlating with reduced patient survival. Combinations of molecules mutated in OSCC may increase and sustain YAP1 activation to the point of oncogenicity. Strikingly, siRNA or pharmacological inhibition of YAP1 blocks murine OSCC onset in vitro and in vivo. Our work justifies targeting YAP1 as therapy for OSCC and perhaps HNSCC, and our mouse model represents a powerful tool for evaluating these agents.

Hypoxia-inducible factor-1α promotes cell survival during ammonia stress response in ovarian cancer stem-like cells.

Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival.

Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway that Is Cell-Density Dependent and Hippo Independent.

Contact inhibition of proliferation is critical for tissue organization, and its dysregulation contributes to tumorigenesis. Merlin/NF2 is a tumor suppressor that governs contact inhibition. Although Merlin/NF2 inhibits YAP1 and TAZ, which are paralogous Hippo pathway transcriptional co-activators and oncoproteins, it is not fully understood how Merlin/NF2-mediated signal transduction triggered by cell-cell contact exerts tumor suppression. Here, we identify Lin28B, an inhibitor of let-7 microRNAs (miRNAs), as an important downstream target of Merlin/NF2. Functional studies revealed that, at low cell density, Merlin/NF2 is phosphorylated and does not bind to Lin28B, allowing Lin28B to enter the nucleus, bind to pri-let-7 miRNAs, and inhibit their maturation in a YAP1/TAZ-independent manner. This inhibition of pri-let-7 maturation then promotes cell growth. However, cell-cell contact triggers Merlin/NF2 dephosphorylation, which sequesters Lin28B in the cytoplasm and permits pri-let-7 maturation. Our results reveal that Merlin/NF2-mediated signaling drives a tumor-suppressive pathway that is cell-density dependent and Hippo independent.

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

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-ß)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-ß pathway may be effective treatment for cHC-CCs and ICCs.

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.

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.

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 G(1) 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.

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 ß-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized ß-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 ß-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.

Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification.

A commonly accepted model of Wnt/ß-catenin signaling involves target gene activation by a complex of ß-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves ß-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

Frodo links Dishevelled to the p120-catenin/Kaiso pathway: distinct catenin subfamilies promote Wnt signals.

p120-catenin is an Arm repeat protein that interacts with varied components such as cadherin, small G proteins, kinases, and the Kaiso transcriptional repressor. Despite recent advances in understanding the roles that p120-catenin and Kaiso play in downstream modulation of Wnt/beta-catenin signaling, the identity of the upstream regulators of the p120-catenin/Kaiso pathway have remained unclear. Here, we find that p120-catenin binds Frodo, which itself interacts with the Wnt pathway protein Dishevelled (Dsh). In Xenopus laevis, we demonstrate that Wnt signals result in Frodo-mediated stabilization of p120-catenin, which, in turn, promotes Kaiso sequestration or removal from the nucleus. Our results point to Dsh and Frodo as upstream regulators of the p120-catenin/Kaiso signaling pathway. Importantly, this suggests that Wnt signals acting through Dsh regulate the stability of p120-catenin in addition to that of beta-catenin, and that each catenin promotes its respective signal in parallel to regulate distinct, as well as shared, direct downstream gene targets.

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity.

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.

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.

Role of crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis.

The Xenopus gene crescent encodes a member of the secreted Frizzled-related protein (sFRP) family and is expressed in the head organizer region. However, the target and function of Crescent in early development are not well understood. Here, we describe a role of Crescent in the regulation of convergent extension movements (CEMs) during gastrulation and neurulation. We show that overexpression of Crescent in whole embryos or animal caps inhibits CEMs without affecting tissue specification. Consistent with this, Crescent efficiently forms complexes with Xwnt11 and Xwnt5a, in contrast to another sFRP, Frzb1. As expected, the inhibitory effect of Crescent or Xwnt11 on CEMs is cancelled when both proteins are coexpressed in the neuroectoderm. Interestingly, when coexpressed in the dorsal mesoderm, the activity of Xwnt11 is rather enhanced by Crescent. Supporting this finding, the inhibition of CEMs by Crescent in mesodermalized but not neuralized animal caps is reversed by the dominant-negative form of Cdc42, a putative mediator of Wnt/Ca2+ pathway. Antisense morpholino oligos for Crescent impair neural plate closure and elicit microcephalic embryos with a shortened trunk without affecting early tissue specification. These data suggest a potential role for Crescent in head formation by regulating a non-canonical Wnt pathway positively in the adjacent posterior mesoderm and negatively in the overlying anterior neuroectoderm.

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 beta-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.

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, beta-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 beta-catenin signaling. Embryos depleted of either gene showed a decreased transcriptional response to TCF3-VP16, a beta-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 beta-catenin.

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.

Molecular link in the sequential induction of the Spemann organizer: direct activation of the cerberus gene by Xlim-1, Xotx2, Mix.1, and Siamois, immediately downstream from Nodal and Wnt signaling.

To elucidate the molecular basis of organizer functions in Xenopus, we sought the target genes of the LIM homeodomain protein Xlim-1, which is one of the organizer-specific transcriptional activators. We found that an activated form of Xlim-1, Xlim-1/3m, initiates ectopic expression of the head-inducing organizer factor gene cerberus in animal caps. Thus, we analyzed the cerberus promoter using reporter assays. We show that three consecutive TAAT motifs of the homeodomain-binding sites between positions -141 and -118, collectively designated the "3xTAAT element," are crucial for the response of the cerberus promoter to Xlim-1/3m, and for its activation in the dorsal region of the embryo. Because cooperative activation of the cerberus promoter by Xnr1 and Xwnt8 also requires the 3xTAAT element, we focused on homeodomain transcriptional activators downstream from either Nodal or Wnt signaling. We found that wild-type Xlim-1 synergistically activates the cerberus promoter with Mix.1 and Siamois through the 3xTAAT element, and this synergy requires the LIM domains of Xlim-1. In contrast, Xotx2 acts synergistically with Mix.1 and Siamois through the TAATCT sequence at -95. Electrophoretic mobility shift assays revealed that Xlim-1, Siamois, and Mix.1 are likely to bind as a complex, in a LIM domain-dependent manner, to the region containing the 3xTAAT element. These data suggest that cerberus is a direct target for Xlim-1, Mix.1, Siamois, and Xotx2. Therefore, we propose a model for the molecular link in the inductive sequence from the formation of the organizer to anterior neural induction.