Dihydroartemisinin promotes ABCG5/G8-mediated cholesterol efflux to potentiate anti-PD-1 therapy through YAP1-TEAD1 complex disruption in hepatocellular carcinoma.

BACKGROUND: Cholesterol accumulation influences the efficacy of immune checkpoint inhibitors (ICIs) in hepatocellular carcinoma (HCC), highlighting a targetable metabolic vulnerability. Dihydroartemisinin (DHA) has shown antitumor activity in HCC; however, its role in regulating cholesterol accumulation remains unclear.

PURPOSE: The purpose of this study is to explore whether DHA enhances anti-PD-1 immunotherapy in HCC by promoting cholesterol efflux and to elucidate the underlying molecular mechanism.

METHODS: A Hepa1-6 subcutaneous tumor-bearing mouse model was established to assess the combined effect of DHA and anti-PD-1 treatment. Multiplex immunohistochemistry was performed to evaluate cytotoxic T-cell infiltration and activation in the tumor microenvironment (TME). Patient-derived organoids (PDOs) were established to evaluate the antitumor activity of DHA. The regulatory mechanism of YAP1/TEAD1 on ABCG5/G8 transcription was explored using chromatin immunoprecipitation (ChIP) qPCR, dual-luciferase reporter assays, and functional gain/loss experiments. DEN/TCPOBOP-induced primary liver tumor model with hepatocyte-specific Yap1 deletion was used to assess the effect of YAP1 deficiency on cholesterol efflux. The direct effect of DHA on the YAP1-TEAD1 interaction was assessed through co-immunoprecipitation (Co-IP) and immunofluorescence (IF) co-localization, and surface plasmon resonance (SPR) analysis.

RESULTS: DHA significantly potentiates anti-PD-1 therapy in HCC-bearing mice, accompanied by reduced serum and intratumoral total cholesterol (TC) levels and increased CD8/granzyme B double-positive signals in tumor tissues, without significant body weight loss. In parallel, ABCG5/G8-mediated cholesterol efflux reduced cholesterol accumulation and suppressed tumor growth, while enhancing cytotoxic immune activity in the TME. Mechanistically, the YAP1-TEAD1 complex directly binds to the promoter regions of ABCG5 and ABCG8 and represses their transcription, thereby limiting cholesterol efflux and promoting intracellular cholesterol accumulation in HCC cells. DHA disrupted the YAP1-TEAD1 interaction, as further supported by SPR analysis, downregulated YAP1 and TEAD1, and restored ABCG5/G8-mediated cholesterol efflux. Notably, DHA-induced ABCG5/ABCG8 upregulation, cholesterol efflux, and antitumor effects were markedly attenuated by YAP1 inhibition, supporting a YAP1-dependent mechanism. In addition, DHA suppressed the growth and viability of organoids derived from patients with HCC.

CONCLUSION: This study reveals that DHA disrupts the YAP1-TEAD1 interaction to relieve transcriptional repression of ABCG5/G8, thereby promoting cholesterol efflux, remodeling the tumor immune microenvironment toward enhanced cytotoxic T-cell activity, and enhancing anti-PD-1 immunotherapy in HCC. These findings provide a novel mechanism for DHA as an immunomodulatory agent and support its clinical translation in combination with ICIs for HCC treatment.