Wnt/β-catenin signaling is a conserved pathway crucially governing development, tissue homeostasis and oncogenesis in metazoan. Through screening, we identified a deubiquitinase (DUB) USP10 as a novel modulator of Wnt/β-catenin signaling. Mechanistically, USP10 binds to Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination. And in parallel, USP10 tethers Axin1 and β-catenin physically, via stabilizing the phase separation of Axin1 through its intrinsically-disordered regions, which is regardless of its enzymatic activity. Functionally, we show USP10 prominently regulates zebrafish embryonic development and murine intestinal homeostasis by antagonizing Wnt/β-catenin signaling. Additionally in human colorectal cancer, USP10 substantially represses cancer growth and correlates with Wnt/β-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes through repressing Wnt/β-catenin signaling and unearthed a novel DUB-dependent and -independent dual-regulating mechanism by which USP10 utilizes in Wnt regulation context-dependently. Our study also suggested the potential of USP10 inhibitor in treating Wnt-related diseases.

Frizzled (Fzd) proteins are Wnt receptors and play essential roles in development, homeostasis, and oncogenesis. How Wnt/Fzd signaling is coupled to physiological regulation remains unknown. Cholesterol is reported as a signaling molecule regulating morphogen such as Hedgehog signaling. Despite the elusiveness of the in‐depth mechanism, it is well‐established that pancreatic cancer specially requires abnormal cholesterol metabolism levels for growth. In this study, it is unexpectedly found that among ten Fzds, Fzd5 has a unique capacity to bind cholesterol specifically through its conserved extracellular linker region. Cholesterol‐binding enables Fzd5 palmitoylation, which is indispensable for receptor maturation and trafficking to the plasma membrane. In Wnt‐addicted pancreatic ductal adenocarcinoma (PDAC), cholesterol stimulates tumor growth via Fzd5‐mediated Wnt/β‐catenin signaling. A natural oxysterol, 25‐hydroxylsterol competes with cholesterol and inhibits Fzd5 maturation and Wnt signaling, thereby alleviating PDAC growth. This cholesterol‐receptor interaction and ensuing receptor lipidation uncover a novel mechanism by which Fzd5 acts as a cholesterol sensor and pivotal connection coupling lipid metabolism to morphogen signaling. These findings further suggest that cholesterol‐targeting may provide new therapeutic opportunities for treating Wnt‐dependent cancers.