AF6 knockout-induced upregulation of bile acid production promotes CXCL14-mediated antitumor immunity in HCC.

[BACKGROUND & AIMS] Bile acid (BA) metabolism plays an important role in the progression of liver cancer; however, the underlying mechanisms remain poorly understood. Hepatic afadin (AF6) is involved in primary BA synthesis, but its role in shaping the tumor microenvironment and immune responses in hepatocellular carcinoma (HCC) is unclear. We hypothesized that an axis involving primary BAs, butyric acid, and CXCL14 contributes to immune regulation in HCC and may inform the development of improved therapeutic strategies.

[METHODS] A diethylnitrosamine-induced liver cancer model with Af6 knockout was established to investigate alterations in BA synthesis. Hepatocyte-specific Af6 deficiency was used to evaluate BA profiles, gut microbiota composition, and microbial metabolites in HCC models. Mechanistic studies were conducted using murine and patient-derived HCC organoids, combined 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 tumor microenvironment via enterohepatic circulation, upregulating Cxcl14 expression and secretion. Cxcl14 recruited activated dendritic cells, 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 bile acid (BA)-driven, microbiota-dependent butyrate-CXCL14 axis. This axis highlights a novel mechanism linking BA metabolism, gut microbiota, and immune regulation, providing potential therapeutic targets for liver cancer treatment. Notably, we describe the first mouse model in which enhanced primary BA production occurs without concomitant alterations in epithelial morphology. This model could provide a valuable platform for investigating BA-associated pathological processes in the gastrointestinal tract, including cancer progression.