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Background Circular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential. Methods To investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation. Results CircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo . Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the “destruction complex” of the Wnt pathway. Released β-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression. Conclusion CircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.

Deregulated Wnt/β-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of-function mutation of CTNNB1, which encodes β-catenin, and loss-of-function mutation of AXIN1 occur in approximately 35% of human HCC samples. Human HCCs with activation of the Wnt/β-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt/β-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Therefore, strategies targeting Wnt/β-catenin have been pursued as possible therapeutics against HCC. Here, we review the genetic alterations and oncogenic roles of aberrant Wnt/β-catenin signaling during hepatocarcinogenesis. In addition, we discuss the implication of this pathway in HCC diagnosis, classification, and personalized treatment.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction. Gammons et al. describe how the clustering of the Wnt receptor complex in clathrin-coated locales allows Axin and GSK3 to dock at adjacent tails of the LRP6 co-receptor to assemble a signalosome that transduces the Wnt signal to the nucleus.

Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells.

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling. Axin is a scaffolding protein known for its role in Wnt signalling that can be marked with a variety of post-translational modifications. Here, Cong et al. demonstrate that USP7 de-ubiquinates Axin and that canonical Wnt signaling output can be increased with USP7 inhibitors.

The colonic epithelial turnover is driven by crypt-base stem cells that express the R-spondin receptor Lgr5. Signals that regulate epithelial regeneration upon stem cell injury are largely unknown. Here, we explore the dynamics of Wnt signaling in the colon. We identify two populations of cells with active Wnt signaling: highly proliferative Lgr5 + /Axin2 + cells, as well as secretory Lgr5 − /Axin2 + cells. Upon Lgr5 + cell depletion, these cells are recruited to contribute to crypt regeneration. Chemical injury induced by DSS leads to a loss of both Lgr5 + cells and Axin2 + cells and epithelial regeneration is driven by Axin2 −  cells, including differentiated Krt20 + surface enterocytes. Regeneration requires stromal Rspo3, which is present at increased levels upon injury and reprograms Lgr5 − but Lgr4 + differentiated cells. In contrast, depletion of stromal Rspo3 impairs crypt regeneration, even upon mild injury. We demonstrate that Rspo3 is essential for epithelial repair via induction of Wnt signaling in differentiated cells. Epithelial turnover in the colon requires stem cells in the crypt that express the R-spondin receptor Lgr5. Here, the authors show that regeneration after colon injury involving loss of Lgr5 + and Axin2 + cells requires stromal derived Rspo3-dependent reprogramming of Lgr4 + differentiated cells, including Krt20 + enterocytes.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative donai analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/ß-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

Axin is one of two essential scaffolds in the canonical Wnt pathway that converts signals at the plasma membrane to signals inhibiting the degradation of β-catenin, leading to its accumulation and specific gene activation. In vertebrates, there are two forms of Axin, Axin1 and Axin2, which are similar at the protein level and genetically redundant. We show here that differential regulation of the two genes on the transcriptional and proteostatic level confers differential responsiveness that can be used in tissue-specific regulation. Such subtle features may distinguish other redundant gene pairs that are commonly found in vertebrates through gene knockout experiments.

Cancer-associated point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth through formation of nonamyloid nanoaggregates that rewire the Axin interactome. Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.