Wnt Ligand Secretion Research Articles

WLS inhibits melanoma cell proliferation through the β-catenin signalling pathway and induces spontaneous metastasis.

Elevated levels of nuclear β-catenin are associated with higher rates of survival in patients with melanoma, raising questions as to how ß-catenin is regulated in this context. In the present study, we investigated the formal possibility that the secretion of WNT ligands that stabilize ß-catenin may be regulated in melanoma and thus contributes to differences in ß-catenin levels. We find that WLS, a conserved transmembrane protein necessary for WNT secretion, is decreased in both melanoma cell lines and in patient tumours relative to skin and to benign nevi. Unexpectedly, reducing endogenous WLS with shRNAs in human melanoma cell lines promotes spontaneous lung metastasis in xenografts in mice and promotes cell proliferation in vitro. Conversely, overexpression of WLS inhibits cell proliferation in vitro. Activating β-catenin downstream of WNT secretion blocks the increased cell migration and proliferation observed in the presence of WLS shRNAs, while inhibiting WNT signalling rescues the growth defects induced by excess WLS. These data suggest that WLS functions as a negative regulator of melanoma proliferation and spontaneous metastasis by activating WNT/β-catenin signalling.

Wls Is Expressed in the Epidermis and Regulates Embryonic Hair Follicle Induction in Mice

Wnt proteins are secreted molecules that play multiple roles during hair follicle development and postnatal hair cycling. Wntless (Wls) is a cargo protein required for the secretion of various Wnt ligands. However, its role during hair follicle development and hair cycling remains unclear. Here, we examined the expression of Wls during hair follicle induction and postnatal hair cycling. We also conditionally deleted Wls with K14-cre to investigate its role in hair follicle induction. K14-cre;Wlsc/c mice exhibited abnormal hair follicle development, which is possibly caused by impaired canonical Wnt signaling. Meanwhile, Wnt5a is also expressed in embryonic epidermis, but Wnt5a null mice showed no significant defect in embryonic hair follicle morphogenesis. Therefore, Wls may regulate hair follicle induction by mediating the Wnt/β-catenin pathway.

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

PD08-11: Targeting Porcupine, a Critical Node for Wnt Signalling in Cancer.

Abstract Wnt ligands were first discovered based upon their transforming activity in the setting of mouse mammary tumor virus (MMTV) induced mammary carcinogenesis. Wnt signalling is required for normal development of the mammary gland and dysregulated Wnt signalling is implicated in cancers of the breast and colon among others. Furthermore, lobular breast cancer commonly shows reduced expression of E-cadherin that can lead to release of membrane-bound b-catenin into the cytoplasm and potentially increase Wnt signalling in the presence of Wnt ligand. To date, however, low-molecular weight therapeutic inhibitors of the Wnt pathway have not been developed. We set out to discover key points of intervention in the Wnt pathway and to develop low molecular weight inhibitors against such nodes. To this end, a cellular high-throughput screen for small molecule inhibitors of the Wnt pathway was performed. In this assay, cells secreting Wnt3a were co-cultured with cells harboring a luciferase reporter driven by the Wnt/beta-catenin responsive elements. Potent and specific Wnt pathway inhibitors were identified from this screen. To discern the mechanism of action of these compounds, a lead inhibitor was radiolabeled and used to probe protein targets in a radioligand binding assay. From these assays a candidate target of the inhibitor was identified as Porcupine. Porcupine is a membrane bound O-acyltransferase enzyme that is required for and dedicated to palmitoylating Wnt ligands, a necessary step in the process of Wnt ligand secretion. Inhibition of Porcupine blocks Wnt dependent activities, including LRP6 phosphorylation and the expression of Wnt target genes, such as Axin2, which in turn reduces the growth of cancer cells dependent on autocrine or paracrine Wnt signaling. A selective and orally bioavailable Porcupine inhibitor (NVP-LGK974) that inhibits the Wnt pathway in vitro and in vivo was developed through medicinal chemistry optimization. In preclinical evaluation, the compound robustly suppressed Wnt pathway signaling in vivo resulting in tumor regression in a murine breast cancer model driven by MMTV-Wnt1. In addition, this inhibitor attenuated tumor growth as a single agent and induced tumor regression in combination with Taxol in a human primary breast tumor model. Wnt signalling is also important for the maintenance and homeostasis of normal tissues including gastrointestinal tissue. However, preclinical rodent efficacy models of cancer demonstrate that intermittent pharmacological inhibition of Porcupine can be effective against the tumor and mostly spare the normal gastrointestinal tissue, with an acceptable therapeutic window. A Phase I dose escalation study of NVP-LGK974 in patients with melanoma and lobular breast cancer to evaluate the safety, tolerability, PK, and PD properties is expected to begin this year. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-11.

P24 proteins are required for secretion of Wnt ligands

During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.

Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome

The Drosophila porcupine gene is required for secretion of wingless and other Wnt proteins, and sporadic mutations in its unique human ortholog, PORCN, cause a pleiotropic X-linked dominant disorder, focal dermal hypoplasia (FDH, also known as Goltz syndrome). We generated a conditional allele of the X-linked mouse Porcn gene and analyzed its requirement in Wnt signaling and embryonic development. We find that Porcn-deficient cells exhibit a cell-autonomous defect in Wnt ligand secretion but remain responsive to exogenous Wnts. Consistent with the female-specific inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phenotype previously associated with loss of Wnt activity. Heterozygous Porcn mutant females exhibit a spectrum of limb, skin, and body patterning abnormalities resembling those observed in human patients with FDH. Many of these defects are recapitulated by ectoderm-specific deletion of Porcn, substantiating a long-standing hypothesis regarding the etiology of human FDH and extending previous studies that have focused on downstream elements of Wnt signaling, such as β-catenin. Conditional deletion of Porcn thus provides an experimental model of FDH, as well as a valuable tool to probe Wnt ligand function in vivo.

A Wnt-ow of Opportunity: Targeting the Wnt/β-Catenin Pathway in Breast Cancer

Aberrant activation of the Wnt/beta-catenin signaling pathway is a hallmark of many tumors, including breast cancer. In the normal breast, tightly regulated expression of Wnt/beta-catenin pathway components, including Wnts and the APC tumor suppressor, dictates its role in balancing stem cell self-renewal, maintenance and differentiation during embryonic and postnatal development. Therefore, not surprisingly, dysregulation of Wnt/beta-catenin signaling through overexpression of pathway activators, such as Wnts or stabilized beta-catenin, or targeted disruption of inhibitors, such as APC, leads to mammary tumorigenesis in several genetically engineered mouse (GEM) models. These models are powerful tools to dissect the importance of Wnt/beta-catenin signaling in human breast cancer because they recapitulate some of the histological features of human breast cancers that demonstrate pathway dysregulation. Over the last decade, numerous approaches have been developed to target the Wnt/beta-catenin pathway in tumor cells, from antagonizing Wnt ligand secretion or binding to promoting beta-catenin degradation to specifically blocking beta-catenin-mediated transcriptional activity. Despite sizeable hurdles because of gaps in our knowledge of most efficacious ways to inhibit the pathway, the breast cancer subtypes to target and how pathway antagonists might be used in combination therapy, crippling Wnt/beta-catenin signaling offers a tremendous opportunity to impact breast cancer pathogenesis. This review will provide an overview of the current understanding of Wnt/beta-catenin pathway involvement in regulating normal breast development and morphogenesis, the generation of Wnt/beta-catenin-dependent GEM models of human breast cancer, upregulation of signaling in human breast cancers and the compelling therapeutic strategies aimed at targeting the Wnt/beta-catenin pathway that show promising anti-tumor activity.

Potent Antineoplastic Activity of Two Inhibitors of Lymphoid Enhancer Binding Factor-1 (LEF-1) in Chronic Lymphocytic Leukemia (B-CLL).

Abstract 885Recent studies indicate factors governing aberrant activation of WNT signaling in chronic lymphoctic leukemia (CLL) cells. Thus, there is an increased secretion of WNT ligands indicating an autocrine loop leading to the extended survival of CLL cells. Lymphoid enhancer binding factor-1 (LEF-1) is a potent transcription factor regulating the expression of several WNT induced target genes. A comprehensive gene expression profiling from two independent studies revealed that LEF-1 mRNA was ∼3,000 fold overexpressed in B-CLL when compared to its healthy counterpart. Hence LEF-1 is a transcription factor expressed exclusively in CLL cells.The objective of this present study is to demonstrate the therapeutic benefit of inhibiting LEF-1 expression in B-CLL cells using novel small molecule inhibitors CGP049090 and PKF115-584 in vivo and in vitro. JVM-3 cells and primary CLL cells were investigated by siRNA mediated knock down of LEF-1 and viability was assayed after 16h of incubation by flow cytometry. In vitro cytotoxicity and IC50 of the two compounds was enumerated using ATP based cell viability assay. Apoptotic response was investigated in time course experiments. Specificity of the small molecules was demonstrated by co-immunoprecipitation experiments for the LEF-1/βcatenin interaction in primary CLL cells. In vivo efficacy of the small molecules inhibitors were studied using a JVM-3 subcutaneous xenograft model in nu/nu mice.The results indicate there is a high protein expression and nuclear localization of LEF-1 and β-catenin indicating active LEF-1 mediated transcription in CLL cells, whereas LEF-1 remained undetectable in healthy B cells. Preliminary experiments of LEF-1 inhibition using siRNAs resulted in increased apoptosis indicating LEF-1 to be an important player in the survival of B-CLL cells. This observation was extended using CGP049090 and PKF-115584 as they induce both dose and time dependent cytotoxicity in B-CLL, whereas healthy B cells are not significantly affected. The IC50 for CGP049090 and PKF-115584 in CLL cells were 0.7 μM and 0.9 μM, respectively. Healthy B cells were not significantly affected, as ascertained by the fact that IC50 values could not be reached due to lacking total cell death. CGP049090 and PKF-115584 induced apoptotic cell death in primary CLL cells and cell lines by cleavage of caspases 8, 9, 3 and 7 and subsequent cleavage of poly (adenosine diphospate-ribose) polymerase (PARP). Both the inhibitors also altered the expression of several anti apoptotic proteins like XIAP, mcl-1 and bcl-2. Co-immunoprecipitation experiments revealed that both the inhibitors effectively break the β-catenin/LEF-1 interaction, resulting in down regulation of expression of LEF-1 target genes such as c-myc, cyclin D1 and LEF-1. Furthermore, the inhibitors were tested in an in vivo JVM-3 subcutaneous xenograft nude mouse model resulted in >70% inhibition of tumor growth and an increase in the median survival of the treated group without leading to systemic toxicity. Immunohistochemistry analysis of the tumor sections revealed LEF-1 down regulation paralleled by inhibition of proliferation by down regulation of Proliferating Cell Nuclear Antigen (PCNA) and increase in apoptosis (induction of cleaved PARP). In summary, we show that LEF-1 is a potential therapeutic target in the treatment of CLL. Both CGP049090 and PKF115-584 show potent inhibitory effects on the survival of CLL cells in vitro and in vivo without affecting healthy B-cells, suggesting them as potential anti-cancer agents in CLL and other neoplastic malignancies with aberrant LEF-1/TCF transcriptional activity. Further investigations are warranted to determine the feasibility of these small molecules for therapeutic approaches in humans. Disclosures:Schlösser:Novartis: Employment. Schmitt:Novartis: Employment. Hallek:BayerScheringAG: Honoraria, Research Funding.

Xenopus Wntless and the retromer complex cooperate to regulate XWnt4 secretion.

Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multipass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila melanogaster embryogenesis. In this study, we show that Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus. Moreover, the Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.

Smed-Evi/Wntless is required for β-catenin-dependent and-independent processes during planarian regeneration

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-beta-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-beta-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through beta-catenin-dependent and -independent pathways.