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A modular optogenetic method for higher-order protein oligomerization uses a single cryptochrome 2-based fusion for rapid, reversible and tunable oligomerization in response to blue light. Inducible aggregation can be used to specifically activate different signaling pathways. We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.

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.

Tumor-associated macrophages (TAMs) are a major component of tumor microenvironment (TME) and play pivotal roles in the progression of hepatocellular carcinoma (HCC). Wnt signaling is evolutionarily conserved and participates in liver tumorigenesis. Several studies have shown that macrophage-derived Wnt ligands can activate Wnt signaling in tumor cells. However, whether Wnt ligands secreted by tumor cells can trigger Wnt signaling in macrophages is still elusive. In this study, we first verified that canonical Wnt/β-catenin signaling was activated during monocyte-to-macrophage differentiation and in M2-polarized macrophages. Knockdown of β-catenin in M2 macrophages exhibited stronger antitumor characteristics when cocultured with Hepa1-6 HCC cells in a series of experiments. Activation of Wnt signaling promoted M2 macrophage polarization through c-Myc. Moreover, co-culturing naïve macrophages with Hepa1-6 HCC cells in which Wnt ligands secretion was blocked by knockdown of Wntless inhibited M2 polarization in vitro. Consistently, the growth of HCC tumor orthotopically inoculated with Wntless-silenced Hepa1-6 cells was impeded, and the phenotype of M2-like TAMs was abrogated due to attenuated Wnt/β-catenin signaling in TAMs, leading to subverted immunosuppressive TME. Finally, we confirmed the correlation between M2 macrophage polarization and nuclear β-catenin accumulation in CD68 + macrophages in human HCC biopsies. Taken together, our study indicates that tumor cells-derived Wnt ligands stimulate M2-like polarization of TAMs via canonical Wnt/β-catenin signaling, which results in tumor growth, migration, metastasis, and immunosuppression in HCC. To block Wnts secretion from tumor cells and/or Wnt/β-catenin signal activation in TAMs may be potential strategy for HCC therapy in future.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid–liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2—a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis. Esposito et al. show that TGF-β-induced DACT1 forms biomolecular condensates that sequester CK2 to repress Wnt signalling and modulate bone metastasis in cancer.

ten Berge and colleagues show that Wnt signalling is required for self-renewal of embryonic stem cells through inhibition of their differentiation into epiblast stem cells. Together, Wnt and LIF signalling can support derivation and self-renewal of embryonic stem cells. Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref.  1 ). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks 2 . In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types 3 , 4 , 5 , 6 . The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A β-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling. The transmembrane protein Wntless (WLS) is essential for the secretion of Wnt proteins, which are signaling molecules of the Wnt signaling pathways. Here, the authors present the 2.2 Å cryo-EM structure of human WLS in complex with Wnt3a and discuss mechanistic implications for the palmitoleoylation of Wnt3a that is required for WLS mediated Wnt secretion.

Tumor‐associated macrophages have important roles in hepatocellular carcinoma (HCC) initiation and progression. Long noncoding RNAs (lncRNAs) have also been reported to be involved in HCC. In this study, we explored how lncRNA LINC00662 may influence HCC progression through both tumor cell‐dependent and macrophage‐dependent mechanisms. LINC00662 was found to be upregulated in HCC, and high LINC00662 levels correlated with poor survival of HCC patients. LINC00662 upregulated WNT3A expression and secretion via competitively binding miR‐15a, miR‐16, and miR‐107. Through inducing WNT3A secretion, LINC00662 activated Wnt/β‐catenin signaling in HCC cells in an autocrine manner and further promoted HCC cell proliferation, cell cycle, and tumor cell invasion, while repressing HCC cell apoptosis. In addition, acting through WNT3A secretion, LINC00662 activated Wnt/β‐catenin signaling in macrophages in a paracrine manner and further promoted M2 macrophage polarization. Via activating Wnt/β‐catenin signaling and M2 macrophages polarization, LINC00662 significantly promoted HCC tumor growth and metastasis in vivo. Hence, targeting LINC00662 may provide novel therapeutic strategy against HCC. Schematic model of LINC00662 functions. LINC00662 upregulates WNT3A expression and secretion via competitively binding miR‐15a/16/107. Secreted WNT3A activates Wnt/β‐catenin in HCC cells in an autocrine manner and promotes HCC cell proliferation and invasion. Secreted WNT3A also activates Wnt/β‐catenin in macrophages in a paracrine manner and promotes M2 macrophage polarization. Finally, LINC00662 promotes HCC tumor growth and metastasis via upregulating WNT3A.

Abstract The Wnt/β-catenin pathway initiates a signaling cascade that is critical in cell differentiation and the normal development of multiple organ systems. The reactivation of this pathway has been documented in experimental and human idiopathic pulmonary fibrosis, wherein Wnt/β-catenin activation has been implicated in epithelial-cell repair. Furthermore, the canonical ligand Wnt3a is known to induce myofibroblast differentiation; however, the role of noncanonical Wnt ligands remains unclear. This study showed significantly higher levels of Wnt11 expression in cells from both patients with idiopathic pulmonary fibrosis and bleomycin-treated mice, as well as in TGFβ-treated mouse lung fibroblasts. Moreover, Wnt11 induced myofibroblast differentiation as manifested by increased α-SMA (ACTA2) expression, which was similar to that induced by canonical Wnt3a/β-catenin signaling. Further investigation revealed that Wnt11 induction of α-SMA was associated with the activation of JNK (c-Jun N-terminal kinase)/c-Jun signaling and was inhibited by a JNK inhibitor. The potential importance of this signaling pathway was supported by in vivo evidence showing significantly increased levels of Wnt11 and activated JNK in the lungs of mice with bleomycin-induced pulmonary fibrosis. Interestingly, fibroblasts did not express canonical Wnt3a, but treatment of these cells with exogenous Wnt3a induced endogenous Wnt11 and Wnt5a, resulting in repression of the Wnt3a/β-catenin target gene Axin2. These findings suggested that the noncanonical Wnt induction of myofibroblast differentiation mediated by the JNK/c-Jun pathway might play a significant role in pulmonary fibrosis, in addition to or in synergy with canonical Wnt3a/β-catenin signaling. Moreover, Wnt3a activation of noncanonical Wnt signaling might trigger a switch from canonical to noncanonical Wnt signaling to induce myofibroblast differentiation.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/β-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in β-catenin ( CTNNB1 ) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper ( Piper nigrum ) and long pepper ( Piper longum ). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/β-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of β-catenin, β-catenin S33A or dnTCF4 VP16, while also suppressing β-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. β-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that β-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, β-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of β-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease. Aberrant Wnt/b-catenin signaling is thought to be a major driver of hepatocellular carcinoma. Here, the authors show that β-Catenin is predominantly integrated within the AJ complex during the early stages of this cancer and enhance EGFR signaling to promote tumour survival.