Abstract
<title>Abstract</title> <p>Dysregulation of glycosphingolipid (GSL) metabolism contributes to cancer progression by altering membrane organization, signaling pathways, and cellular adaptation. Sulfated glycosphingolipids (Sulfo-GSLs), including sulfated lactosylceramide, are elevated in several malignancies and have been implicated in tumor cell adhesion and metastasis. We previously identified elevated Sulfo-GSL levels in hepatocellular carcinoma (HCC) HepG2 cells, suggesting that targeting GSL biosynthesis may represent a potential therapeutic strategy. Here, we investigated the molecular consequences of UDP-glucose ceramide glucosyltransferase (UGCG) inhibition by eliglustat in HepG2 by integrating MALDI mass spectrometry-based lipid profiling with RNA sequencing. Eliglustat treatment induced dose-dependent remodeling of the cellular lipid profile, characterized by marked reduction of Sulfo-GSL species and a concomitant increase in selected phosphatidylinositol species. Lipidomic analysis revealed differential sensitivity of Sulfo-GSL molecular species, with preferential depletion of non-hydroxylated forms and relative preservation of hydroxylated species, indicating distinct metabolic responses within the same lipid class. Transcriptomic changes were limited at 20 µM eliglustat, whereas 30 µM induced extensive transcriptional reprogramming involving activation of immediate-early response genes, including EGR, FOS/JUN, and KLF family members, together with enrichment of inflammatory, stress-response, and senescence-associated pathways. Conversely, pathways related to cell-cycle progression, DNA replication, and chromatin organization were suppressed. Genes involved in GSL metabolism were also modulated, suggesting activation of compensatory mechanisms to restore lipid homeostasis. These findings demonstrate that UGCG inhibition induces coordinated lipidomic and transcriptional remodeling in HepG2, revealing both tumor-suppressive molecular features and adaptive responses to metabolic stress providing new insights into the cellular consequences of targeting GSL metabolism in HCC.</p>