Salvianolic acid B targets MST1/2-YAP axis to drive phospho-Smad3L/C conversion against hepatocarcinogenesis

Background: Hepatocellular carcinoma (HCC), a major cause of Cancer death, lacks effective targeted therapies. Salvianolic acid B (Sal B), a bioactive phytochemical from Salvia miltiorrhiza, exhibits anti-HCC activity; however, its molecular mechanisms remain incompletely defined. Critically, the interaction between Hippo effector YAP and tumor-suppressive COOH-terminally phosphorylated SMAD3 (pSmad3C) or oncogenic linker-phosphorylated SMAD3 (pSmad3L) in HCC pathogenesis is unexplored.

Purpose: This study investigates the novel crosstalk between YAP and pSmad3C/3 L as a mechanistic target for Sal B, aiming to elucidate a new anti-HCC strategy.

Methods: TCGA database, molecular docking, and co-immunoprecipitation (Co-IP) assays characterized YAP/pSmad3C-L interaction. In vivo, hepatic fibrosis was induced in MST1/2 DKO (MST1⁻^/⁻; MST2^fl/fl; Alb-Cre) and SMAD3 C-terminal phosphorylation knock in (pSmad3C⁺^/⁻) mice using DEN/CCl₄/EtOH (DCE), followed by HCC induction with or without Sal B intervention. In vitro, HepG2 models with YAP modulation (overexpression/shRNA knockdown) and SMAD3 variant transfection (WT, EPSM, 3S-A) complemented mechanistic studies.

Results: A direct interaction between the YAP and pSmad3C/3 L was identified. Sal B's efficacy was abolished in MST1/2 DKO or pSmad3C^± mice, although pathological manifestations associated with pSmad3C knockdown were more severe, MST1/2-YAP emerged as Sal B's dominant targeted in vivo. Furthermore, YAP knockdown and pSmad3C overexpression intensifies Sal B's anti-HCC effect by promoting a shift from pro-tumorigenic pSmad3L/PAI-1//c-Myc signaling to tumor-suppressive pSmad3C/p21 signaling and activation of MST1/2; conversely, upregulation of YAP and pSmad3L reversed this effect in vitro.

Conclusion: Sal B impedes HCC by targeting MST1/2-YAP axis to drive pSmad3L/C conversion. This work identifies the first phytochemical strategy against HCC that reprograms YAP/pSmad3C-L interactions, positioning MST1/2-YAP-driven phospho-Smad3 conversion as a promising therapeutic axis.

Hepatocellular carcinoma; MST1/2-YAP; Phospho-Smad3 conversion; Salvianolic acid B; Signaling crosstalk; pSmad3C/L.