Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.     

Polyethylenimine-cationized β-catenin protein transduction activates the Wnt canonical signaling pathway more effectively than cationic lipid-based transduction

The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by β-catenin protein transduction. Constitutively active β-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active β-catenin protein was added to HEK-293 cells, and induction of several Wnt/β-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active β-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active β-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.     

Mesd is a general inhibitor of different Wnt ligands in Wnt/LRP signaling and inhibits PC-3 tumor growth in vivo

Mesd is a specialized chaperone for Wnt co-receptor low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6, which contain four β-propeller/epidermal growth factor modules, named E1 to E4 from N- to C-terminal, in their extracellular domains. Herein, we demonstrated that recombinant Mesd protein is a general Wnt inhibitor that blocks Wnt/β-catenin signaling induced not only by LRP6 E1-E2-binding Wnts but also by LRP6 E3-E4-binding Wnts. We also found that Mesd suppressed Wnt/β-catenin signaling induced by Wnt1 in prostate cancer PC-3 cells, and inhibited tumor growth in PC-3 xenograft model. Our results indicate that Mesd is a universal inhibitor of Wnt/LRP signaling on the cell surface.     

Both LRP5 and LRP6 receptors are required to respond to physiological Wnt ligands in mammary epithelial cells and fibroblasts

A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.     

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

The Wnt Co-Receptor Lrp6 Is Required for Normal Mouse Mammary Gland Development

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.     

Developmental Stage and Time Dictate the Fate of Wnt/β-Catenin-Responsive Stem Cells in the Mammary Gland

The mammary epithelium undergoes extensive growth and remodeling during pregnancy, suggesting a role for stem cells. Yet their origin, identity, and behavior in the intact tissue remain unknown. Using an Axin2(CreERT2) allele, we labeled and traced Wnt/β-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/β-catenin signaling around birth and shows that, depending on the developmental stage, Axin2(+)?cells contribute differently to basal and luminal epithelial cell lineages of the mammary epithelium. Moreover, an important difference exists between the developmental potential tested in transplantation assays and that displayed by the same cell population in?situ. Finally, Axin2(+) cells in the adult build alveolar structures during multiple pregnancies, demonstrating the existence of a Wnt/β-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/β-catenin signaling in the mammary epithelium and offers insights into the developmental fate of mammary gland stem and progenitor cells.     

Kallistatin antagonizes Wnt/β-catenin signaling and cancer cell motility via binding to low-density lipoprotein receptor-related protein 6

Kallistatin, a plasma protein, exerts pleiotropic effects in inhibiting angiogenesis, inflammation and tumor growth. Canonical Wnt signaling is the primary pathway for oncogenesis in the mammary gland. In this study, we demonstrate that kallistatin bound to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), thus, blocking Wnt/β-catenin signaling and Wnt-mediated growth and migration in MDA-MB-231 breast cancer cells. Kallistatin inhibited Wnt3a-induced proliferation, migration, and invasion of cultured breast cancer cells. Moreover, kallistatin was bound to LRP6 in breast cancer cells, as identified by immunoprecipitation followed by western blot. Kallistatin suppressed Wnt3a-mediated phosphorylation of LRP6 and glycogen synthase kinase-3β, and the elevation of cytosolic β-catenin levels. Furthermore, kallistatin antagonized Wnt3a-induced expression of c-Myc, cyclin D1, and vascular endothelial growth factor. These findings indicate a novel role of kallistatin in preventing breast tumor growth and mobility by direct interaction with LRP6, leading to blockade of the canonical Wnt signaling pathway.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

Inducible expression of Wnt genes during adult hepatic stem/progenitor cell response

In injured livers where hepatocyte growth is severely limited, facultative hepatic stem/progenitor cells, termed oval cells in rodents, are known to emerge and contribute to the regeneration process. Here, we investigated a possible involvement of Wnt signaling during mouse oval cell response and found significant upregulation of several Wnt genes including Wnt7a, Wnt7b, and Wnt10a. Accordingly, increase of β-catenin protein was observed in oval cell compartments. Pharmacological activation of the canonical Wnt/β-catenin signaling induced proliferation of cultured hepatic stem/progenitor cell lines. These results together implicate the role of Wnt/β-catenin signaling in adult hepatic stem/progenitor cell response.