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Wnt3a, one of Wnt family members, plays key roles in regulating pleiotropic cellular functions, including self‐renewal, proliferation, differentiation, and motility. Accumulating evidence has suggested that Wnt3a promotes or suppresses tumor progression via the canonical Wnt signaling pathway depending on cancer type. In addition, the roles of Wnt3a signaling can be inhibited by multiple proteins or chemicals. Herein, we summarize the latest findings on Wnt3a as an important therapeutic target in cancer.

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.

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.

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.

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.

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.

AICP is a crucial process that maintaining tissue homeostasis and regeneration. In the past, cell death was perceived merely as a means to discard cells without functional consequences. However, during regeneration, effector caspases orchestrate apoptosis, releasing signals that activate stem cells, thereby compensating for tissue loss across various animal models. Despite significant progress, the activation of Wnt3a by caspase-3 remains a focal point of research gaps in AICP mechanisms, spanning from lower to higher regenerative animals. This inquiry into the molecular intricacies of caspase-3-induced Wnt3a activation contributes to a deeper understanding of the links between regeneration and cancer mechanisms. Our report provides current updates on AICP pathways, delineating research gaps and highlighting the potential for future investigations aimed at enhancing our comprehension of this intricate process.

The paired box 6 ( Pax6 ) gene encodes a highly conserved transcription factor, involved in the development of eyes, brain, and endocrine glands. Homozygous loss of Pax6 resulted in neonatal death in mice, plus loss of eyes and malformation of cerebral cortex. In patients with heterozygous Pax6 mutations, a reduction in thickness of the frontoparietal cortex was detected, which was also observed in small eye mice. In this study, we found that Pax6 overexpression increased the cortical thickness, especially in the intermediate zone of the cortex, which conflicts with the report of Manuel et al. Pax6 overexpression appears to detain neurons in the intermediate zone while promoting cell proliferation. It is worth noting that the impact of Pax6 overexpression on cortical thickness and neuronal migration was temporal, explaining the differences with other reports. We postulated that the alteration of Pax6 isoform ratio by autoregulation might be responsible for this. JASPAR analysis together with the results of qPCR, Western blot, CUT&Tag, and rescue experiments revealed that Pax6 regulates neuronal migration and cell proliferation by indirectly mediating Wnt3a expression. Therefore, we propose that Pax6 participates in corticogenesis via interaction with Wnt3a in regulating neuronal migration and cell proliferation.

The Wnt signaling pathway plays a critical role in initiation, progression, invasion and metastasis of cancer. Wnt3a as a canonical Wnt ligand is strongly implicated in the etiology and pathology of a number of diseases including cancer. Depending on cancer type, Wnt3a enhances or suppresses metastasis, cell proliferation and apoptosis of cancer cells. This review summarizes the role of Wnt3a in the pathogenesis of different cancers including colorectal, prostate, hepatocellular, lung and leukemia, for promoting greater understanding and clinical management of these diseases.

Hepatocellular carcinoma (HCC) has a poor prognosis and high mortality. Ferroptosis, an iron‐dependent regulated cell death process, is implicated in cancer development and treatment. Wnt signaling and lysyl oxidase (Lox) family members are associated with ferroptosis. This study investigates how Wnt3a and/or Loxl2 knockdown affects liver cancer stem cells (LCSCs) and orthotopic tumor growth in mice, and explores the role of ferroptosis‐related genes. Bioinformatics identified ferroptosis‐ and HCC‐associated differentially expressed genes (DEGs) correlated with Wnt3a/Loxl2. LCSCs sorted from Hep3B were transduced with lentivirus for gene knockdown. Ferroptosis markers and DEG expression were analyzed. Wnt3a/Loxl2 knockout mice were generated using CRISPR‐Cas9, and orthotopic tumor models were established. Tumor inhibition rates, ferroptosis‐related indicators, and DEG expression were assessed. 199 ferroptosis‐related DEGs were identified in HCC; ZEB1 was selected as a key gene via PPI analysis. Wnt3a/Loxl2 knockdown increased Fe2+ and MDA, and decreased GSH, most evidently in double‐knockdown cells. In vivo, single‐ and double‐knockout groups showed suppressed tumor growth, with inhibition rates of 51%, 71%, and 93%, respectively. Tumor tissues exhibited similar ferroptosis marker changes. ZEB1 was upregulated in both cellular and animal knockout models. Wnt3a/Loxl2 knockdown promotes ferroptosis in LCSCs and inhibits orthotopic tumor growth, with the strongest effect following dual‐gene knockout. ZEB1 may be an important regulatory factor in this process. Using bioinformatic analysis, LCSC models, and transgenic mice, we demonstrate that co‐inhibition of Wnt3a and Loxl2 potently triggers ferroptosis and inhibits orthotopic liver tumorigenesis. The enhanced antitumor effect upon dual targeting, mediated through ZEB1 upregulation, highlights a promising combinatory approach against HCC.