Abstract

Background & Aims Bile acid (BA) metabolism has been found to play an important role in the progression of liver cancer, but the specific mechanism is poorly understood. Hepatic afadin (AF6) is implicated in primary BA synthesis, but its role in modulating the tumor microenvironment (TME) and immune response in hepatocellular carcinoma (HCC) is poorly understood. We expect that an axis incorporating primary BAs, butyric acid, and CXCL14 will facilitate the development of improved clinical therapies for the treatment of patients with HCC. Methods We developed a DEN-induced liver cancer model with Af6 knockout to study BA synthesis alterations. Hepatocyte-specific Af6 deficiency in HCC models was used to assess BA levels, gut microbiota composition, and microbial metabolites. Mechanistic interrogation combined murine and patient-derived HCC organoids with single-cell and bulk RNA sequencing. Results Af6 deficiency increased primary BA levels, altering gut microbiota composition and elevating butyrate production. Butyrate, not BAs, reshaped the TME via enterohepatic circulation, upregulating Cxcl14 expression and secretion. Cxcl14 recruited activated dendritic cells (DCs), enhancing CD8 + T-cell cytotoxicity. Hepatocyte-specific Cxcl14 overexpression created a tumor-suppressive immune microenvironment, significantly inhibiting HCC progression. Conclusions AF6 modulates primary BA synthesis, driving gut microbiota-dependent butyrate production and Cxcl14-mediated immune remodeling in HCC. This axis highlights a novel mechanism linking BA metabolism, gut microbiota, and immune regulation, offering potential therapeutic targets for liver cancer treatment. Impact and implications This study establishes that hepatic Af6 deficiency restrains hepatocarcinogenesis via a BA-driven, microbiota-dependent butyrate-CXCL14 axis. This axis highlights a novel mechanism linking B