Sailing with the Wnt: Charting the Wnt processing and secretion route

Wnt proteins are members of a highly conserved family of signalling molecules that play a central role in development and disease. During the past years, the different signalling pathways that are triggered by Wnt proteins have been studied in detail, but it is still largely unknown how a functional Wnt protein is produced and secreted. The recent finding that Wnt proteins are post-translationally modified and the discovery of the Wnt binding protein Wntless and its trafficking by the retromer complex show that Wnt secretion is a complex and highly regulated process. In this review, we will give an overview of the Wnt maturation and secretion pathway and discuss how this process may influence the spreading and signalling activity of Wnt.     

Regulation of Wnt protein secretion and its role in gradient formation

In metazoans, many developmental and disease-related processes are mediated by Wnt proteins, which are secreted by specific cells to regulate cellular programmes in the surrounding tissue. Although the Wnt-induced signal-transduction cascades are well studied, little is known about how Wnts are secreted. The discovery of Porcupine, an endoplasmic-reticulum-resident acyltransferase, led to closer inspection of the secretory routes of Wnts, and the analysis of Wnt secretion has become an exciting new area of research. Wnt post-translational modifications, interaction partners and subcellular localizations now indicate that Wnt release is tightly regulated. In this review, we summarize recent advances in the field of Wnt secretion and discuss the possibility that separate pathways might regulate the release of lipid-linked morphogens for short-range and long-range signalling.     

WNT secretion and signalling in human disease

Wnt signalling, a key pathway involved in various aspects of embryonic development, also underlies many human diseases, in particular, cancer. Research focused on signal transduction within signal-receiving cells led to the discovery of many Wnt pathway components, but study of the secretion of Wnt ligands themselves was neglected until recently. Attention was drawn to this highly regulated process by the association of aberrant Wnt levels with an increasing number of diseases. Studying the biogenesis and processing of active Wnt ligands will open new avenues for generating therapeutics to specifically target aberrant Wnt signalling. Here we review the proteins required for Wnt secretion and signalling at the plasma membrane, ending with a discussion on potential therapeutic approaches to treat Wnt-induced diseases.     

Updating the Wnt pathways

In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases.     

Adult mammalian stem cells: the role of Wnt, Lgr5 and R-spondins

After its discovery as oncogen and morphogen, studies on Wnt focused initially on its role in animal development. With the finding that the colorectal tumour suppressor gene APC is a negative regulator of the Wnt pathway in (colorectal) cancer, attention gradually shifted to the study of the role of Wnt signalling in the adult. The first indication that adult Wnt signalling controls stem cells came from a Tcf4 knockout experiment: mutant mice failed to build crypt stem cell compartments. This observation was followed by similar findings in multiple other tissues. Recent studies have indicated that Wnt agonists of the R-spondin family provide potent growth stimuli for crypts in vivo and in vitro. Independently, Lgr5 was found as an exquisite marker for these crypt stem cells. The story has come full circle with the finding that the stem cell marker Lgr5 constitutes the receptor for R-spondins and occurs in complex with Frizzled/Lrp.     

Regulation of Wnt signalling by receptor-mediated endocytosis

Wnts compromise a large family of secreted and hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. Recent advances in the field of Wnt signalling have revealed that Wnt activates multiple intracellular cascades, resulting in the regulation of cellular proliferation, differentiation, migration and polarity. However, it is not clear how Wnt activates these pathways after it binds to the receptors. It has been shown that Wnt and its antagonist Dickkopf are internalized with their receptors. This review highlights distinct endocytic pathways correlate with specificity of Wnt signalling events.     

Wnt3a promotes radioresistance via autophagy in squamous cell carcinoma of the head and neck

The canonical Wnt/β‐catenin signalling pathway and autophagy play critical roles in cancer progression. However, the role of Wnt‐mediated autophagy in cancer radioresistance remains unclear. In this study, we found that irradiation activated the Wnt/β‐catenin and autophagic signalling pathways in squamous cell carcinoma of the head and neck (SCCHN). Wnt3a is a classical ligand that activated the Wnt/β‐catenin signalling pathway, induced autophagy and decreased the sensitivity of SCCHN to irradiation both in vitro and in vivo. Further mechanistic analysis revealed that Wnt3a promoted SCCHN radioresistance via protective autophagy. Finally, expression of the Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients showing high expression of Wnt3a displayed a worse prognosis. Taken together, our study indicates that both the canonical Wnt and autophagic signalling pathways are valuable targets for sensitizing SCCHN to irradiation.     

Wise,a context-dependent activator and inhibitor of Wnt signalling

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.     

Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.     

Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed

Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.     

Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension

Differentiation onset in the vertebrate body axis is controlled by a conserved switch from fibroblast growth factor (FGF) to retinoid signalling, which is also apparent in the extending limb and aberrant in many cancer cell lines. FGF protects tail-end stem zone cells from precocious differentiation by inhibiting retinoid synthesis, whereas later-produced retinoic acid (RA) attenuates FGF signalling and drives differentiation. The timing of RA production is therefore crucial for the preservation of stem zone cells and the continued extension of the body axis. Here we show that canonical Wnt signalling mediates the transition from FGF to retinoid signalling in the newly generated chick body axis. FGF promotes Wnt8c expression, which persists in the neuroepithelium as FGF signalling declines. Wnt signals then act here to repress neuronal differentiation. Furthermore, although FGF inhibition of neuronal differentiation involves repression of the RA-responsive gene, retinoic acid receptor beta (RARbeta), Wnt signals are weaker repressors of neuron production and do not interfere with RA signal transduction. Strikingly, as FGF signals decline in the extending axis, Wnt signals now elicit RA synthesis in neighbouring presomitic mesoderm. This study identifies a directional signalling relay that leads from FGF to retinoid signalling and demonstrates that Wnt signals serve, as cells leave the stem zone, to permit and promote RA activity, providing a mechanism to control the timing of the FGF-RA differentiation switch.     

From individual Wnt pathways towards a Wnt signalling network

Wnt proteins play important roles during vertebrate and invertebrate development. They obviously have the ability to activate different intracellular signalling pathways. Based on the characteristic intracellular mediators used, these are commonly described as the Wnt/beta-catenin, the Wnt/calcium and the Wnt/Jun N-terminal kinase pathways (also called planar cell polarity pathway). In the past, these different signalling events were mainly described as individual and independent signalling branches. Here, we discuss the possibility that Wnt proteins activate a complex intracellular signalling network rather than individual pathways and suggest a graph representation of this network. Furthermore, we discuss different ways of how to predict the specific outcome of an activation of this network in a particular cell type, which will require the use of mathematical models. We point out that the use of deterministic approaches via the application of differential equations is suitable to model only small aspects of the whole network and that more qualitative approaches are possibly a suitable starting point for the prediction of the global behaviour of such large protein interaction networks.     

Mitotic and mitogenic Wnt signalling

Canonical Wnt signalling plays an important role in development, tissue homeostasis, and cancer. At the cellular level, canonical Wnt signalling acts by regulating cell fate, cell growth, and cell proliferation. With regard to proliferation, there is increasing evidence for a complex interaction between canonical Wnt signalling and the cell cycle. Mitogenic Wnt signalling regulates cell proliferation by promoting G1 phase. In mitosis, components of the Wnt signalling cascade function directly in spindle formation. Moreover, Wnt signalling is strongly activated in mitosis, suggesting that 'mitotic Wnt signalling' plays an important role to orchestrate a cell division program. Here, we review the complex interplay between Wnt signalling and the cell cycle.     

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.     

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. We analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). We show that simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. We identify Tc-Arr as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation. We discuss how Wnt signalling is regulated at the receptor and co-receptor levels in a dose-dependent fashion.     

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.