Canonical Wnt Research Articles

β-Catenin–related protein WRM-1 is a multifunctional regulatory subunit of the LIT-1 MAPK complex

Vertebrate β-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four β-catenin-related proteins present in the round worm Caenorhabditis elegans. The fourth β-catenin-related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription of Wnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein. We showed previously that WRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate β-catenin.

Cadherin 11, a miR-675 Target, Induces N-Cadherin Expression and Epithelial–Mesenchymal Transition in Melasma

Cadherin 11 (CDH11) was identified as a target of miR-675 by using a luciferase reporter assay. CDH11 expression and miR-675 expression were inversely correlated. CDH11 expression was not detected in melanocytes, but CDH11 expression in fibroblasts and keratinocytes positively influenced melanogenesis via the canonical Wnt and AKT activation pathways in cocultured melanocytes. CDH11 in fibroblasts or keratinocytes induced N-cadherin and Twist1 expression, while decreasing E-cadherin expression. This suggests a role for CDH11 in epithelial-mesenchymal transition. CDH11 in fibroblasts also induced the migration of cocultured melanocytes. N-cadherin knockdown abolished the tyrosinase expression that was induced in CDH11-overexpressing fibroblasts. Collectively, our data indicate that CDH11 in fibroblasts and keratinocytes is a target of miR-675, and could be involved in melanogenesis through the induction of N-cadherin during epithelial-mesenchymal transition.

Regeneration of insulin-producing pancreatic cells using a volatile bioactive compound and human teeth

Transplantation of insulin (INS)-secreting cells differentiated in vitro from stem cells promises a safer and easier treatment of severe diabetes mellitus. A volatile bioactive compound, hydrogen sulfide (H2S), promotes cell differentiation; human tooth-pulp stem cells undergo hepatic differentiation. The aim of this study is to develop a novel protocol using H2S to enhance pancreatic differentiation from the CD117+ cell fraction of human tooth pulp. During the differentiation, the cells were exposed to 0.1 ng ml−1 H2S. Immunocytochemistry, RT-PCR, determination of INS c-peptide content and flow cytometry of pancreatically related markers were carried out. Expression of WNT and the PI3K/AKT signaling pathway were also determined by PCR array. After differentiation, INS, glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PPY) were positive when examined by immunofluorescence. INS and GCG were also determined flow-cytometrically. Only the cells expressing INS increased after H2S exposure. The number of cells expressing GCG was significantly decreased. Genes involved in canonical WNT and the WNT/calcium pathways were highly expressed after H2S exposure. H2S accelerated INS synthesis and secretion by regenerated INS-producing cells from human teeth. All signaling pathway functions of the PI3K-AKT pathway were extremely activated by H2S exposure. The matured INS-producing cells originating in human teeth were increased by H2S in order to control blood-glucose level.

Abstract 5448: PTK7 as a potential therapeutic target in ovarian cancer

Abstract The identification of proteins that selectively discriminate between tumor cells and normal adult cells allows for the specific targeting of diseased cells with antibody therapeutics. One such recently identified protein, PTK7, is an onco-fetal membrane protein which exhibits limited expression and function in adults. PTK7 was identified as a member of the RTK super family but lacks a functional kinase domain. Normally, PTK7 is expressed early in development and its loss is associated with severe defects in neural tube closure and sensory hair cell bundle formation. Functionally, little is known about the signaling involving PTK7, but it has been linked to both the canonical and noncanonical WNT pathways. Recently, PTK7 expression has been shown to be upregulated in a number of cancers including: ovarian, melanoma, leukemia, lung, pancreatic, colon, renal and breast. In vitro and in vivo studies support a role in regulating angiogenesis, invasion & survival. To further validate PTK7 as a potential cancer target that may be required for tumor maintenance and progression, we analyzed the expression of PTK7 in normal and tumor samples, and validated an in vitro and in vivo role of PTK7 on cell growth in ovarian cancer cell lines using both genetic tools and polyclonal antibodies. Silencing PTK7 with stably expressed inducible shRNAs is shown to inhibit the growth of ovarian cancer cell lines in vitro and to lead to delayed tumor growth upon PTK7 knockdown in murine tumor xenograft models. Further supporting the role of PTK7 as a potential antibody target, polyclonal antibodies to PTK7 are shown to inhibit the growth of SKOV3 and OVCAR8 cells in vitro. Although 4 human antibodies derived from phage display failed to inhibit in vitro cell growth, these results suggest that functionally blocking PTK7 may lead to the inhibition of ovarian tumor growth and is a potential target for antibody therapies. Citation Format: Zhihu Ding, Amanda Lennon, Keli Perron, David Harper, Hui Su, Meredith Wolfram, Joshua Murtie, Stuart Licht, Jason Pinckney, Helene Simonds-Mannes, Kimberly Bishop, Julie-Ann Gavigan, Dinesh Bangari, Maureen Magnay, William Weber, David Reczek, William Brondyk, Vicky Drewett, Marc Trombe, Dietmar Hoffmann, Raffaele Baffa, Serena Silver, Victoria Richon, Christopher Winter, Venkat Reddy, Richard C. Gregory. PTK7 as a potential therapeutic target in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2014-5448

Abstract 3009: Membrane pseudokinase PTK7 regulates metastasis

Abstract Studies by others indicate that the membrane pseudokinase 7 (PTK7) regulates canonical and non-canonical Wnt pathways and controls orientation of cells in a tissue plane (planar cell polarity, PCP). There is still no clear understanding of the PTK7 role in cancer: Is it pro-tumorigenic or anti-tumorigenic? Our live cell imaging and cell invasion studies suggest that full-length PTK7 expressed in HT1080 fibrosarcoma cells inhibits directed cell motility, lamellipodia polarization and cell invasion. Using the fluorescence imaging we demonstrated that the expression of full-length PTK7 in HT1080-RFP cells in nude mice significantly reduces lung colonization (metastasis). The pro-invasive membrane type-1 matrix metalloprotease (MT1-MMP/MMP-14) coupled with an ADAM/ADAMTS family member(s) directly cleave PTK7 at the cell surface and reverse the inhibitory anti-invasive signal of full-length PTK7. Ectodomain proteolysis was a prerequisite for the intramembrane cleavage of the residual fragments of PTK7 by γ-secretase. The resulting C-terminal PTK7 fragment translocated to the cell nucleus. To identify the molecular mechanisms downstream of PTK7 proteolysis, we used genome-wide transcriptional profiling and kinome analysis. Full-length PTK7 and the PTK7 mutants representing the cleaved PTK7 forms differentially regulated the expression of multiple genes and induced signaling pathways associated with cell movement, Wnt signaling and cancer. We believe that the regulation of PTK7 proteolysis opens new possibilities to control Wnt signaling, cancer cell invasion and metastasis. Citation Format: Vladislav Golubkov, Yong Zhang, Natalie Prigozhina, Robert M. Hoffman, Alex Strongin. Membrane pseudokinase PTK7 regulates metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2014-3009

The cross-talk between canonical and non-canonical Wnt-dependent pathways regulates P-glycoprotein expression in human blood-brain barrier cells.

In this work, we investigate if and how transducers of the 'canonical' Wnt pathway, i.e., Wnt/glycogen synthase kinase 3 (GSK3)/β-catenin, and transducers of the 'non-canonical' Wnt pathway, i.e., Wnt/RhoA/RhoA kinase (RhoAK), cooperate to control the expression of P-glycoprotein (Pgp) in blood-brain barrier (BBB) cells. By analyzing human primary brain microvascular endothelial cells constitutively activated for RhoA, silenced for RhoA or treated with the RhoAK inhibitor Y27632, we found that RhoAK phosphorylated and activated the protein tyrosine phosphatase 1B (PTP1B), which dephosphorylated tyrosine 216 of GSK3, decreasing the GSK3-mediated inhibition of β-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of β-catenin, and reduced the β-catenin-driven transcription of Pgp. The RhoAK inhibition increased the delivery of Pgp substrates like doxorubicin across the BBB and improved the doxorubicin efficacy against glioblastoma cells co-cultured under a BBB monolayer. Our data demonstrate that in human BBB cells the expression of Pgp is controlled by a cross-talk between canonical and non-canonical Wnt pathways. The disruption of this cross-talk, e.g., by inhibiting RhoAK, downregulates Pgp and increases the delivery of Pgp substrates across the BBB.

An unexpected role for a Wnt-inhibitor: Dickkopf-1 triggers a novel cancer survival mechanism through modulation of aldehyde-dehydrogenase-1 activity

It is widely accepted that canonical Wnt (cWnt) signaling is required for the differentiation of osteoprogenitors into osteoblasts. Furthermore, tumor-derived secretion of the cWnt-antagonist Dickkopf-1 (Dkk-1) is known to cause bone destruction, inhibition of repair and metastasis in many bone malignancies, but its role in osteosarcoma (OS) is still under debate. In this study, we examined the role of Dkk-1in OS by engineering its overexpression in the osteochondral sarcoma line MOS-J. Consistent with the known role of Dkk-1 in osteoblast differentiation, Dkk-1 inhibited osteogenesis by the MOSJ cells themselves and also in surrounding tissue when implanted in vivo. Surprisingly, Dkk-1 also had unexpected effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro and caused the formation of larger and more destructive tumors than controls upon orthotopic implantation. These effects were attributed in part to upregulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1). Direct inhibition of ALDH1 reduced viability under stressful culture conditions, whereas pharmacological inhibition of cWnt or overexpression of ALDH1 had a protective effect. Furthermore, we observed that ALDH1 was transcriptionally activated in a c-Jun-dependent manner through a pathway consisting of RhoA, MAP-kinase-kinase-4 and Jun N-terminal Kinase (JNK), indicating that noncanonical planar cell polarity-like Wnt signaling was the mechanism responsible. Together, our results therefore demonstrate that Dkk-1 enhances resistance of OS cells to stress by tipping the balance of Wnt signaling in favor of the non-canonical Jun-mediated Wnt pathways. In turn, this results in transcriptional activation of ALDH1 through Jun-responsive promoter elements. This is the first report linking Dkk-1 to tumor stress resistance, further supporting the targeting of Dkk-1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells.

STLV-1-infected Japanese macaque as a model of HTLV-1 infection

sively sequencing the provirus integration sites. We found that some clones proliferated distinctively in monkeys with higher proviral load. T lymphocytes expressing Tax in the peripheral blood were largely CD4+. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain, indicating that STLV-1 is oncogenic in Japanese macaques. We also assessed the molecular function of STLV-1 Tax and STLV-1 bZIP factor (SBZ). STLV-1 Tax activated NFAT, AP-1, canonical Wnt and canonical NF-kappa B pathways, whereas SBZ suppressed those signaling pathways. SBZ enhanced TGF-beta signaling, but STLV-1 Tax suppressed it. These findings suggest that STLV-1 Tax and SBZ have similar functions to their counterparts of HTLV-1. In addition, we found that administration of anti-CCR4 antibody, which is currently used in Japan for the treatment of ATL patients, efficiently reduced proviral load in STLV-1-infected Japanese macaques. Our study provides the evidence that Japanese macaques naturally infected with STLV-1 correspond to HTLV-1 carriers and are a suitable animal model to investigate the pathogenesis of HTLV-1 and novel therapeutic strategies.

Wnt pathway regulation by long-term moderate exercise in rat hippocampus

An active lifestyle involving regular exercise reduces the deleterious effects of the aging process. At the cerebral level, both synaptic plasticity and neurogenesis are modulated by exercise, although the molecular mechanisms underlying these effects are not clearly understood. In the mature nervous system, the canonical Wnt (Wnt/β-catenin) signaling pathway is implicated in neuroprotection and synaptic plasticity. Here, we examined whether the Wnt pathway could be modulated in adult male rat hippocampus by long-term moderate exercise (treadmill running) or enrichment (handling/environmental stimulation). Sedentary animals showed higher protein levels of the Wnt antagonist, Dkk-1, the lowest levels being found in the exercised group. Although there was no evidence of any changes in activation of the LRP6 receptor, the total levels of LRP6 were higher in exercised and enriched animals. Analysis of some of the components implicated in the phosphorylation of β-catenin, which leads ultimately to its proteasomal degradation, revealed higher levels and activation of Axin1 and GSK-3α/β respectively in sedentary animals. However neither different phosphorylated forms nor total β-catenin protein levels differed between the experimental groups. Higher protein levels of Axin2 and the antiapoptotic protein, Bcl-2, were found with exercise and handling, whereas the proapototic, Bax, was unaffected. Thus, our results suggest activation of the Wnt pathway not only with moderate exercise, but also with the handling of the animals.

Cauli: A Mouse Strain with an Ift140 Mutation That Results in a Skeletal Ciliopathy Modelling Jeune Syndrome

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

Rare missense variants in DVL1, one of the human counterparts of the Drosophila dishevelled gene, do not confer increased risk for neural tube defects

Neural tube defects (NTDs) are severe malformations that arise when the neural tube fails to close during embryogenesis. The planar cell polarity pathway is involved in neural tube closure and has been implicated in the pathogenesis of NTDs both in animal models and human cohorts. Dishevelled (Dvl/Dsh) is a multi-module protein and a key regulator of both the canonical Wnt and the PCP pathway. In mouse, all Dvl1(-/-) ; Dvl2(-/-) double mutants display craniorachischisis, a severe form of open NTDs. Recently, we have reported a possible role for rare variants of DVL2 as risk factors for NTDs. In view of these data, we hypothesized that DVL1 mutations might increase the risk for NTDs in some cases. Resequencing of the DVL1 gene in a cohort of 473 NTDs patients and 150 ethnically matched controls was performed. Prediction of the downstream effects of the nonsynonymous variants was done using computational methods. We identified six missense variants that were absent in our ethnically matched controls group, and four of them (p.Arg153Cys; p.Glu544Arg; p.Arg568Trp; p.Val644Phe) were predicted to have a functional effect on protein structure by one or more bioinformatic programs. However, there was no difference in the overall rate of deleterious variants between the patients and controls (four in patients and three in controls; p=0.36). Our findings did not provide evidence for the implication of DVL1 in the pathogenesis of human NTDs.

Regulation of osteoclast differentiation by Wnt signals

Osteoclast differentiation is regulated by receptor activator of NF-κB ligand, colony stimulating factor-1 and osteoprotegerin. These cytokines are derived from osteoblast-lineage cells such as osteoblasts and osteocytes. Wnt has been shown to be critical for osteoblast differentiation through β-catenin dependent canonical and β-catenin independent noncanonical Wnt signals. Recent studies have shown that osteoclastogenesis is also regulated by Wnt signals. This review summarizes recent advances in roles of Wnt signals in bone resorption under physiological and pathological conditions.

Abstract 4005: The human Norrin/beta-catenin signaling enhancer TSPAN12 is expressed on colorectal tumors; evidence for cell-type specific responsiveness to Norrin signaling in the tumor microenvironment.

Abstract Introduction. While the majority of colorectal cancers (CRC) harbor mutations in Wnt pathway component APC, leading to constitutive pathway activation, tumor cells remain responsive to Wnt signals arising at the cell membrane through the interaction of extracellular Wnt ligands and cell surface frizzled (Fz) receptors. The strength of the Wnt signal can be modified through epigenetic modification of genes encoding for extracellular Wnt pathway inhibitors that produce proteins released into the tumor microenvironment. Norrin is an extracellular molecule that binds to Fz4 and induces beta-catenin (canonical Wnt) signaling. This signaling is also dependent on the expression of TSPAN12 which functions as a signal enhancer. Norrin promotes angiogenesis in the eye but the role of Norrin signaling in colorectal cancer is not known. In this study we have examined the expression of Norrin pathway components in CRC and other cell types in the tumor microenvironment. Methods. Human CRC and normal colon tissues, including mucosa, stroma and endothelial cells were analyzed by immunohistochemistry and reversed transcription polymerase chain reaction in order to define expression of Norrin, Fz4 and TSPAN12. In vitro, endothelial cells were grown on collagen in the presence of CRC cell line-produced conditioned medium (CM), with or without Norrin blocking antibody. Results. Norrin and Fz4 were expressed in normal colonic mucosa, CRC, and endothelial cells. TSPAN12, the component that usually determines what tissues will respond to the Norrin signaling, was expressed in CRC but not in normal colonic mucosa. Low amounts of endothelial cell TSPAN12 in tissue sections was difficult to quantitate due to insufficient sensitivity of the method. In vitro, elongation of endothelial cells treated with CM from CRC cell lines was partly abrogated by Norrin blocking antibodies, suggesting responsiveness of these cells to Norrin-mediated signals. Conclusions. Critical components of the Norrin/beta-catenin pathway are expressed in CRC and the colon tumor microenvironment but TSPAN12, the signal enhancer that promotes signaling through this pathway is found on CRC cells and not expressed on normal mucosa In vitro evidence suggests that Norrin signaling also affects endothelial cells. Implications. The distribution of TSPAN expression in the CRC tumor microenvironment, and the effect of Norrin blocking antibodies on CRC CM-treated endothelial cells in vitro, suggest that the predominant roles of Norrin are a direct action on CRC and potentially also promotion of tumor angiogenesis. Citation Format: Kestutis Planutis, Marina Planutiene, Randall F. Holcombe. The human Norrin/beta-catenin signaling enhancer TSPAN12 is expressed on colorectal tumors; evidence for cell-type specific responsiveness to Norrin signaling in the tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4005. doi:10.1158/1538-7445.AM2013-4005

Genotoxic Exposure during Juvenile Growth of Mammary Gland Depletes Stem Cell Activity and Inhibits Wnt Signaling

Various types of somatic stem cell have been tested for their response to genotoxic exposure, since these cells are likely to be important to regeneration, aging and cancer. In this study, we evaluated the response of mammary stem cells to genotoxic exposure during ductal growth in juveniles. Exposure to the polycyclic aromatic hydrocarbon (DMBA; 7,12 dimethylbenz[a]anthracene) had no gross effect on outgrowth and morphogenesis of the ductal tree, or upon lobuloalveolar growth during pregnancy. However, by fat pad assay, we found that mammary stem cell activity was reduced by 80% in glands from adults that were exposed to genotoxins as juveniles. The associated basal cell lineage was depleted. Both basal and luminal cells showed a robust response to genotoxic exposure (including γH2AX phosphorylation, pS15p53 and pT68Chk2), with durable hyperproliferation, but little cytotoxicity. Since the phenotype of these glands (low basal cell fraction, low stem cell activity) phenocopies mammary glands with loss of function for Wnt signaling, we measured Wnt signaling in genotoxin-exposed glands, and found a durable reduction in the activation of the canonical signaling Wnt receptors, Lrp5/6. Furthermore, when mammary epithelial cells were treated with Wnt3a, DMBA exposure reduced the basal cell population and Lrp activation was ablated. We conclude that during active ductal growth, Wnt-dependent mammary stem cells are sensitized to cell death by genotoxin exposure. Our conclusion may be important for other tissues, since all solid tumor stem cell activities have been shown to be Wnt-dependent to date.

Control of osteogenesis by the canonical Wnt and BMP pathways in vivo

Although many regulators of skeletogenesis have been functionally characterized, one current challenge is to integrate this information into regulatory networks. Here, we discuss how the canonical Wnt and Smad-dependent BMP pathways interact together and play antagonistic or cooperative roles at different steps of osteogenesis, in the context of the developing vertebrate embryo. Early on, BMP signaling specifies multipotent mesenchymal cells into osteochondroprogenitors. In turn, the function of Wnt signaling is to drive these osteochondroprogenitors towards an osteoblastic fate. Subsequently, both pathways promote osteoblast differentiation, albeit with notable mechanistic differences. In osteocytes, the ultimate stage of osteogenic differentiation, the Wnt and BMP pathways exert opposite effects on the control of bone resorption by osteoclasts. We describe how the dynamic molecular wiring of the canonical Wnt and Smad-dependent BMP signaling into the skeletal cell genetic programme is critical for the generation of bone-specific cell types during development.

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.

TNF-alpha stimulates alkaline phosphatase and mineralization through canonical and non-canonical WNT signals in human osteoblast progenitors

TNF-alpha stimulates alkaline phosphatase and mineralization through canonical and non-canonical WNT signals in human osteoblast progenitors

Abstract 3240: The Wnt secretion factor Evi/Wls/Gpr177 promotes glioma tumorigenesis

Abstract Malignant astrocytomas are highly aggressive brain tumors with poor prognosis. Despite multimodal therapy, including surgical resection followed by combined radio- and chemotherapy, the median survival of glioblastoma patients has remained as low as 12-14 months throughout the past decade. While a number of structural genomic changes and dysregulation of signaling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Recent evidence supports the involvement of canonical and non-canonical Wnt signaling in glioma development and malignant progression. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signaling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signaling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumors in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signaling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumorigenesis, identifying a pathway- specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3240. doi:1538-7445.AM2012-3240

Microenvironmental Regulation of Cancer Stem Cell Phenotypes

Cancer is a complex set of diseases, driven by genomic instability overlaid with epigenetic modifications. Two prevailing concepts, the stochastic theory and the hierarchical theory, are traditionally used to understand tumor progression. These seemingly contradictory theories can be reconciled with the concept of cellular plasticity, such that certain genetic mutations enable epigenetic alterations in cell fate. A growing body of evidence suggests that cancer cells co-opt embryonic stem cell-associated regulatory networks in order to sustain tumor cell plasticity concomitant with growth and progression. The expression of these stem cell associated factors is regulated by dynamic niches, characterized by cellderived proteins as well as biophysical features such low oxygen tensions. In this review we describe specific embryo-associated proteins such as NODAL, NOTCH, and canonical WNT, which cooperate to maintain stem cell phenotypes in cancer. We also illustrate how biophysical factors, in particular oxygen, can orchestrate plasticity by modulating the expression of stem cell-associated proteins. As the microenvironment is known to play a key role in cellular regulation, it is essential to understand its role in cancer progression in order to improve and create new therapies.

Developmental effects of tobacco smoke exposure during human embryonic stem cell differentiation are mediated through the transforming growth factor-β superfamily member, Nodal

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.