The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6.

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/ß-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

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.

USP47 deubiquitylates Groucho/TLE to promote Wnt-ß-catenin signaling.

The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.

Feedback in the ß-catenin destruction complex imparts bistability and cellular memory.

Wnt ligands are considered classical morphogens, for which the strength of the cellular response is proportional to the concentration of the ligand. Herein, we show an emergent property of bistability arising from feedback among the Wnt destruction complex proteins that target the key transcriptional co-activator ß-catenin for degradation. Using biochemical reconstitution, we identified positive feedback between the scaffold protein Axin and the kinase glycogen synthase kinase 3 (GSK3). Theoretical modeling of this feedback between Axin and GSK3 suggested that the activity of the destruction complex exhibits bistable behavior. We experimentally confirmed these predictions by demonstrating that cellular cytoplasmic ß-catenin concentrations exhibit an "all-or-none" response with sustained memory (hysteresis) of the signaling input. This bistable behavior was transformed into a graded response and memory was lost through inhibition of GSK3. These findings provide a mechanism for establishing decisive, switch-like cellular response and memory upon Wnt pathway stimulation.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

Nuclear Regulation of Wnt/ß-Catenin Signaling: It's a Complex Situation.

Wnt signaling is an evolutionarily conserved metazoan cell communication pathway required for proper animal development. Of the myriad of signaling events that have been ascribed to cellular activation by Wnt ligands, the canonical Wnt/ß-catenin pathway has been the most studied and best understood. Misregulation of Wnt/ß-catenin signaling has been implicated in developmental defects in the embryo and major diseases in the adult. Despite the latter, no drugs that inhibit the Wnt/ß-catenin pathway have been approved by the FDA. In this review, we explore the least understood step in the Wnt/ß-catenin pathway-nuclear regulation of Wnt target gene transcription. We initially describe our current understanding of the importation of ß-catenin into the nucleus. We then focus on the mechanism of action of the major nuclear proteins implicated in driving gene transcription. Finally, we explore the concept of a nuclear Wnt enhanceosome and propose a modified model that describes the necessary components for the transcription of Wnt target genes.

Casein Kinase 1α as a Regulator of Wnt-Driven Cancer.

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.

Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization.

Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. Herein, we tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. We find that the critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease.

Developmental regulation of Wnt signaling by Nagk and the UDP-GlcNAc salvage pathway.

In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFß, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.

A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila.

Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin-a negative regulator of Wnt signaling-and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus, the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. Herein, we tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, we provide genetic and biochemical evidence that Rnf146 and Tnks function in the same proteolysis pathway in vivo Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that, in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.

Wingless/Wnt Signaling in Intestinal Development, Homeostasis, Regeneration and Tumorigenesis: A Drosophila Perspective.

In mammals, the Wnt/ß-catenin signal transduction pathway regulates intestinal stem cell maintenance and proliferation, whereas Wnt pathway hyperactivation, resulting primarily from the inactivation of the tumor suppressor Adenomatous polyposis coli (APC), triggers the development of the vast majority of colorectal cancers. The Drosophila adult gut has recently emerged as a powerful model to elucidate the mechanisms by which Wingless/Wnt signaling regulates intestinal development, homeostasis, regeneration, and tumorigenesis. Herein, we review recent insights on the roles of Wnt signaling in Drosophila intestinal physiology and pathology.

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway.

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting ß-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased ß-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote ß-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC.

The aberrant activation of Wnt signal transduction initiates the development of 90% of colorectal cancers, the majority of which arise from inactivation of the tumor suppressor Adenomatous polyposis coli (APC). In the classical model for Wnt signaling, the primary role of APC is to act, together with the concentration-limiting scaffold protein Axin, in a "destruction complex" that directs the phosphorylation and consequent proteasomal degradation of the transcriptional activator ß-catenin, thereby preventing signaling in the Wnt-off state. Following Wnt stimulation, Axin is recruited to a multiprotein "signalosome" required for pathway activation. Whereas it is well-documented that APC is essential in the destruction complex, APC's role in this complex remains elusive. Here, we demonstrate in Drosophila that Axin exists in two distinct phosphorylation states in Wnt-off and Wnt-on conditions, respectively, that underlie its roles in the destruction complex and signalosome. These two Axin phosphorylation states are catalyzed by glycogen synthase kinase 3 (GSK3), and unexpectedly, completely dependent on APC in both unstimulated and Wnt-stimulated conditions. In a major revision of the classical model, we show that APC is essential not only in the destruction complex, but also for the rapid transition in Axin that occurs after Wnt stimulation and Axin's subsequent association with the Wnt co-receptor LRP6/Arrow, one of the earliest steps in pathway activation. We propose that this novel requirement for APC in Axin regulation through phosphorylation both prevents signaling in the Wnt-off state and promotes signaling immediately following Wnt stimulation.

Intestinal stem cell overproliferation resulting from inactivation of the APC tumor suppressor requires the transcription cofactors Earthbound and Erect wing.

Wnt/ß-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). The ß-catenin-TCF transcription complex activates both the physiological expression of Wnt target genes in the normal intestinal epithelium and their aberrantly increased expression in colorectal tumors. Whether mechanistic differences in the Wnt transcription machinery drive these distinct levels of target gene activation in physiological versus pathological states remains uncertain, but is relevant for the design of new therapeutic strategies. Here, using a Drosophila model, we demonstrate that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers. Together, our findings reveal distinct requirements for Ebd and Ewg in physiological Wnt pathway activation versus oncogenic Wnt pathway hyperactivation following Apc1 loss. Such differentially utilized transcription cofactors may offer new opportunities for the selective targeting of Wnt-driven cancers.

Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling.

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.

Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling.

Deregulation of the Wnt signal transduction pathway underlies numerous congenital disorders and cancers. Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure; however, due to the very low levels of endogenous Axin, this model is based largely on examination of Axin at supraphysiological levels. Here, we analyze the subcellular distribution of endogenous Drosophila Axin in vivo and find that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. We conclude that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

Wnt pathway activation by ADP-ribosylation.

Wnt/ß-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly.

The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila.

Wnt/ß-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer.