Abstract 591: The ubiquitin ligase HUWE1 controls the balance between β-catenin functions in WNT signaling and cell adhesion.

Abstract β-catenin plays two major roles in the cell, one as the key mediator of transcriptional responses to WNT signaling in the cytoplasm and nucleus, and the other as a structural component of adherens junctions, cell-cell adhesion complexes at the plasma membrane. How the balance between these spatially separate and functionally distinct pools of β-catenin is controlled is not well understood. WNT/β-catenin signaling is a fundamental signaling pathway, dysregulation of which can drive many types of cancer. Disassembly of adherens junctions, often caused by reduced β-catenin, is a hallmark of epithelial-mesenchymal transition (EMT), a process that promotes cancer metastasis. The goal of this study was to investigate the mechanisms that regulate the balance between the two spatially and functionally distinct pools of β-catenin. During WNT/β-catenin signaling, the main regulated step is β-catenin phosphorylation and degradation mediated by the destruction complex, composed of the scaffold proteins APC and AXIN1/2, and the kinases casein kinase 1α and GSK3α/β. Using CRISPR, we created two disease model cell lines from haploid human (HAP1) cells: 1. casein kinase 1α knock-out cells, and 2. β-catenin ST-A cells, containing mutations in β-catenin that render it insensitive to phosphorylation and degradation by the destruction complex. In both cell lines, we observed by confocal microscopy that β-catenin accumulates in the cytoplasm and the nucleus, and promotes hyperactive WNT signaling. Importantly, we found that loss of the E3 ubiquitin ligase HUWE1 in casein kinase 1α knock-out cells induced a marked change in the localization of β-catenin from the nucleus to the plasma membrane, which was accompanied by a substantial reduction in WNT/β-catenin signaling. Through proximity ligation assays and a new adherens junction-dependent cell adhesion assay that we developed, we found that localization of β-catenin to the plasma membrane caused by HUWE1 loss promotes β-catenin incorporation into adherens junctions and increases its functional contribution to cell-cell adhesion. In β-catenin ST-A cells that contain non-degradable β-catenin, regulation of β-catenin subcellular localization by HUWE1 was also observed. Taken together, these results demonstrate that HUWE1 regulates the balance between β-catenin transcriptional activity in WNT signaling and its cell adhesion functions. Therefore, regulation of β-catenin functions through HUWE1 may open new therapeutic avenues for cancers caused by hyperactive WNT signaling, and/or in metastasis driven by EMTs. Citation Format: Caleb Kwame Sinclear, Joseph McKenna, Yalan Wu, Praveen Sonkusre, Andres M. Lebensohn. The ubiquitin ligase HUWE1 controls the balance between β-catenin functions in WNT signaling and cell adhesion abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 591.