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.

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.