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.

SELENOP modifies sporadic colorectal carcinogenesis and WNT signaling activity through LRP5/6 interactions.

Although selenium deficiency correlates with colorectal cancer (CRC) risk, the roles of the selenium-rich antioxidant selenoprotein P (SELENOP) in CRC remain unclear. In this study, we defined SELENOP's contributions to sporadic CRC. In human single-cell cRNA-Seq (scRNA-Seq) data sets, we discovered that SELENOP expression rose as normal colon stem cells transformed into adenomas that progressed into carcinomas. We next examined the effects of Selenop KO in a mouse adenoma model that involved conditional, intestinal epithelium-specific deletion of the tumor suppressor adenomatous polyposis coli (Apc) and found that Selenop KO decreased colon tumor incidence and size. We mechanistically interrogated SELENOP-driven phenotypes in tumor organoids as well as in CRC and noncancer cell lines. Selenop-KO tumor organoids demonstrated defects in organoid formation and decreases in WNT target gene expression, which could be reversed by SELENOP restoration. Moreover, SELENOP increased canonical WNT signaling activity in noncancer and CRC cell lines. In defining the mechanism of action of SELENOP, we mapped protein-protein interactions between SELENOP and the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). Last, we confirmed that SELENOP-LRP5/6 interactions contributed to the effects of SELENOP on WNT activity. Overall, our results position SELENOP as a modulator of the WNT signaling pathway in sporadic CRC.

Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic ß-catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling is unknown. Here, we confirm that BVES loss increases ß-catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modeling using a Wnt-driven genetic model and a chemically induced model of colorectal carcinogenesis demonstrate that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.

Phosphorylation of XIAP at threonine 180 controls its activity in Wnt signaling.

X-linked inhibitor of apoptosis (XIAP) plays an important role in preventing apoptotic cell death. XIAP has been shown to participate in signaling pathways, including Wnt signaling. XIAP regulates Wnt signaling by promoting the monoubiquitylation of the co-repressor Groucho/TLE family proteins, decreasing its affinity for the TCF/Lef family of transcription factors and allowing assembly of transcriptionally active ß-catenin-TCF/Lef complexes. We now demonstrate that XIAP is phosphorylated by GSK3 at threonine 180, and that an alanine mutant (XIAPT180A) exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in Xenopus embryos. Although XIAPT180A ubiquitylates TLE3 at wild-type levels in vitro, it exhibits a reduced capacity to ubiquitylate and bind TLE3 in human cells. XIAPT180A binds Smac (also known as DIABLO) and inhibits Fas-induced apoptosis to a similar degree to wild-type XIAP. Our studies uncover a new mechanism by which XIAP is specifically directed towards a Wnt signaling function versus its anti-apoptotic function. These findings have implications for development of anti-XIAP therapeutics for human cancers.

Identification of a Paralog-Specific Notch1 Intracellular Domain Degron.

Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.