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Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43 / ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43 / Znrf3 -mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors. Enhanced Wnt receptor activity is a major cause of cancer development. Here the authors identify camelid single-domain antibody fragments (VHHs) that bind to the Wnt receptor LRP5/6 ectodomain, determine the crystal structures and show that these VHHs selectively inhibit Wnt3- mediated cellular responses and block the growth of mutant Wnt-hypersensitive intestinal tumor organoids.

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6 -deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi -Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling. The low-density lipoprotein receptor-related protein 6 (LRP6) is a co-receptor of the β-catenin-dependent Wnt signaling pathway and interacts with the Wnt inhibitor sclerostin (SOST). Here the authors present the crystal structure of SOST in complex with the LRP6 E1E2 ectodomain construct, which reveals that the SOST C-terminus binds to the LRP6 E2 domain, and further validate this binding site with in vitro and in vivo experiments.

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/μm² to 0.54/μm². Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

The authors describe water-soluble surrogate Wnt agonists, with specificity towards some frizzled (FZD) receptors, which can maintain human intestinal organoid cultures and have effects on the mouse liver in vivo . Water-soluble Wnt agonists Wnt ligands interact with FZDand Lrp5/6-type receptors to influence diverse developmental, homeostatic and pathologic processes through β-catenin-dependent signalling. The promiscuity of Wnt ligands towards several receptors and the fact that Wnts can be hydrophobic make it difficult to produce therapeutic recombinant Wnts. Chris Garcia and colleagues have developed surrogate water-soluble Wnt agonists that have specificity towards certain FZDs.The new agonists act similarly to Wnt3 in differentiation assays towards the osteogenic lineage in vitro , can maintain intestinal organoid cultures, and have in vivo effects on the mouse liver. Elsewhere in this issue, Calvin Kuo and colleagues use these novel water-soluble Wnt agonists in the mouse intestinal stem-cell niche to dissect the respective roles of R-spondin and Wnt ligands, both of which activate similar signalling receptors and pathways. They find that Lgr5 + intestinal stem cells normally differentiate unless both R-spondin and Wnt ligands are present. However, on their own, each ligand acts non-redundantly and in cooperation with Wnt agonists, activating R-spondin receptors to maintain stem-cell competency and these receptors are in turn activated in the presence of R-spondin to drive stem-cell expansion. These water-soluble Wnt agonists could be used in a range of assays to understand this signalling pathway and modulate it in therapeutical applications. Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues 1 . The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors 2 , 3 , 4 . As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

Aggressive behaviours of solid tumours are highly influenced by the tumour microenvironment. Multiple signalling pathways can affect the normal function of stromal fibroblasts in tumours, but how these events are coordinated to generate tumour-promoting cancer-associated fibroblasts (CAFs) is not well understood. Here we show that stromal expression of Dickkopf-3 (DKK3) is associated with aggressive breast, colorectal and ovarian cancers. We demonstrate that DKK3 is a HSF1 effector that modulates the pro-tumorigenic behaviour of CAFs in vitro and in vivo. DKK3 orchestrates a concomitant activation of β-catenin and YAP/TAZ. Whereas β-catenin is dispensable for CAF-mediated ECM remodelling, cancer cell growth and invasion, DKK3-driven YAP/TAZ activation is required to induce tumour-promoting phenotypes. Mechanistically, DKK3 in CAFs acts via canonical Wnt signalling by interfering with the negative regulator Kremen and increasing cell-surface levels of LRP6. This work reveals an unpredicted link between HSF1, Wnt signalling and YAP/TAZ relevant for the generation of tumour-promoting CAFs. It is unclear how specific signalling pathways are coordinated to generate pathologically activated cancer associated fibroblasts (CAFs). Here, Ferrari et al show that stromal expression of Dickkopf-3 (DKK3) associates with aggressive tumours. DKK3 is a HSF-1 effector that activates β-catenin and YAP/TAZ, and DKK3-driven YAP/TAZ activation regulates the pro-tumorigenic behaviour of CAFs.

Wnt signaling plays fundamental roles in organogenesis, tissue regeneration and cancer, but high-resolution structural information of mammalian Wnt proteins is lacking. We solved a 2.8-Å resolution crystal structure of human Wnt3 in complex with mouse Frizzled 8 Cys-rich domain (CRD). Wnt3 grabs the receptor in a manner very similar to that found in Xenopus Wnt8 complexed with the same receptor. Unlike Xenopus Wnt8-bound CRD, however, Wnt3-bound CRD formed a symmetrical dimer in the crystal by exchanging the tip of the unsaturated acyl chain attached to each Wnt3, confirming the ability of Wnt and Frizzled CRD to form a 2:2 complex. The hypervariable ‘linker’ region of Wnt3 formed a β-hairpin protrusion opposite from the Frizzled binding interface, consistent with its proposed role in the coreceptor recognition. Direct binding between this segment and the Wnt coreceptor LRP6 was confirmed, enabling us to build a structural model of the Wnt–Frizzled–LRP6 ternary complex.Takagi and colleagues report the crystal structure of human Wnt3 in complex with the mouse Frizzled 8 Cys-rich domain, a structural model of the Wnt–Frizzled–LRP6 ternary complex and engineered tagged versions of Wnt3a that retain biological function.

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in β-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc , Axin2 and Lef1 . Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced β-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting β-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/β-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/β-catenin signalings during oncogenesis.

Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction. Gammons et al. describe how the clustering of the Wnt receptor complex in clathrin-coated locales allows Axin and GSK3 to dock at adjacent tails of the LRP6 co-receptor to assemble a signalosome that transduces the Wnt signal to the nucleus.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains. Wnt receptors are controlled by their ubiquitin-mediated proteolysis. The authors show that the USP46 deubiquitylase complex potentiates Wnt signaling in human cells, Xenopus , and zebrafish by inhibiting cell surface LRP6 degradation.