Integration of network pharmacology, metabolomics and lipidomics for clarifying the role of sphingolipid metabolism in the treatment of liver cancer by regorafenib

Regorafenib, an FDA-approved drug for advanced primary liver cancer (PLC), could provide survival benefits for patients. However, markers for its therapeutic sensitivity are lacking. This study seeks to identify sensitive targets of regorafenib in PLC from the perspective of small molecular metabolites. Initiated with network pharmacology (NP) to map regorafenib's target landscape and metabolic regulatory network in liver cancer. Subsequently, regorafenib's impact on hepatoma cells was evaluated by flow cytometry, western blotting (WB) and cell viability assay. Advanced metabolomics and lipidomics were employed to elucidate regorafenib's metabolic reprogramming effects in liver cancer. Metabolic enzyme expression was assessed by WB, immunohistochemical and immunofluorescence assays. Ultimately, mendelian randomization (MR) analysis was utilized to investigate the potential causality of sphingolipid metabolism in hepatic cancer. Regorafenib was observed to inhibit hepatoma cell proliferation and cell cycle progression at G0/G1 phase, resulting in significant alterations in sphingolipid levels. It promoted the significant accumulation of 16:0 dihydroceramide (16:0 dhCer) by upregulating ceramide synthase 6 (CERS6) expression and inhibiting dihydroceramide desaturase 1 (DEGS1) activity. The MR analysis revealed that DEGS1 was a risk factor for the development and progression of liver cancer, while cumulative 16:0 dhCer was a protective factor. Sphingolipids, particularly dhCer and regulatory enzymes, may be potential sensitive markers of regorafenib in the treatment of liver cancer, providing new insights for enhancing the treated efficacy of regorafenib in liver cancer. • This is the first report on regorafenib-mediated metabolic changes in liver cancer by metabolomics and lipidomics. • Sphingolipid metabolism plays an important role in the treatment of liver cancer with regorafenib. • Regorafenib induced the accumulation of 16:0 dihydroceramide by regulating DEGS1 and CERS6. • Mendelian randomization analysis revealed the relationship between sphingolipids and hepatic cancer.