AXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. Significance: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.

Bidens pilosa L., a traditional Chinese medicinal herb, has been used in clinical practice for the treatment of inflammatory diseases and cancer. BPA, an extract derived from the whole herb of B. pilosa L., has been shown to possess potent immunomodulatory properties by regulating tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) within the tumor microenvironment (TME) in a mouse syngeneic colorectal cancer (CRC) model. RT-PCR and flow cytometry analyses showed that BPA, together with its flavonoid and polyacetylene constituents, effectively suppressed the differentiation of M2-TAMs and Tregs by downregulating Arg-1 and CD25 expression. They had minimal effects on the expression of markers associated with M1-TAMs and promoted the proliferation of CD4⁺ T cells that were inhibited by M2-TAMs and Tregs. In mice, BPA markedly inhibited the growth of syngeneic CRC tumors, accompanied by decreased serum levels of the immunosuppressive cytokine IL-10 and reduced expression of the proliferative marker Ki67 in tumor tissues. Moreover, BPA downregulated the mRNA expression of markers associated with M2-TAMs and Tregs, while increasing markers associated with M1-TAMs. Western blot analyses of tumor tissues revealed that BPA reduced the expression of marker proteins associated with M2-TAMs and Tregs, while increasing the expression of the immune-stimulatory markers CD80, GITR and CD4. In addition, combined treatment with BPA and 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent for CRC, notably enhanced the anti-tumor effect in mice. These findings indicate that BPA, an active extract of B. pilosa L., showed antitumor activity in mice by suppressing the differentiation of pro-tumorigenic TAMs and Tregs within the TME.

Reported missense mutations in D65_S228 domain of AXIN1

Deleting part of the RGS/APC domain at endogenous levels results in increased β-catenin signaling

Additional analysis of missense variants near the GSK3β domain