Seco-neokadsuranic acid A antagonizes SH3BP1 to suppress hepatocellular carcinoma progression through PPAR pathway activation.

[BACKGROUND] The high mortality of hepatocellular carcinoma (HCC), driven by metastasis and drug resistance, underscores the need for novel therapeutics. The natural triterpenoid seco-neokadsuranic acid A (SA) shows promising antitumor activity; however, its precise molecular targets and mechanisms of action in HCC remain unclear, impeding its clinical development.

[PURPOSE] To interrogate the direct target engagement of SA and establish its mechanistic basis in suppressing HCC metastasis and remodeling antitumor immunity.

[METHODS] An integrated multi-level investigative strategy was utilized. The direct protein target of SA was deconvoluted by human proteome microarray screening coupled with bioinformatic interrogation. The resulting SA-target interaction was rigorously characterized through computational simulations (molecular docking and dynamics), surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA). The anti-HCC efficacy and underlying mechanisms were systematically evaluated in vitro (HepG2 cells) and in vivo (H22 tumor-bearing mice), employing a comprehensive suite of assays to assess proliferation (CCK-8), migration/invasion (Transwell, wound healing), and molecular phenotypes (Western blot, immunofluorescence, immunohistochemistry).

[RESULTS] SH3 domain-binding protein 1 (SH3BP1) was established as a direct target of SA, with clinical data from TIMER, GEPIA2 and UALCAN databases confirming its overexpression in HCC and association with advanced tumor stage and poor survival. Computational simulations characterized the binding stability and identified ARG272 as the critical residue, findings corroborated by SPR and CETSA. Functionally, SA treatment resulted in concentration-related suppression of SH3BP1, inhibition of HCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), as well as disruption of cytoskeletal dynamics and activation of PPAR signaling. In vivo, SA significantly attenuated tumor progression and metastasis, induced apoptosis, and favorably remodeled the immune landscape via enhanced CD4⁺/CD8⁺ T-cell infiltration and suppressed M2 macrophage polarization, all without detectable toxicity. Mechanistic interrogation in SH3BP1-overexpressing models demonstrated that SA rescues metastatic phenotypes and PPAR (peroxisome proliferator-activated receptor) pathway suppression, thereby supporting the existence of a functional SA-SH3BP1-PPAR axis.

[CONCLUSION] This study defines SA as a direct inhibitor of SH3BP1, activating the PPAR pathway and suppressing cytoskeleton-mediated metastasis. Integrated analyses reveal its role in immune remodeling and safety, establishing the SH3BP1-PPAR axis and positioning SA as a promising therapeutic candidate for HCC.