Graph attention network-driven single-cell omics analysis reveals how B4GALNT1-mediated glycosylation remodelling of the ferroptosis-pyroptosis network promotes drug resistance in hepatocellular carcinoma.

BACKGROUND

Acquired resistance to sorafenib in hepatocellular carcinoma (HCC) is accompanied by coordinated changes in oxidative-stress handling and regulated cell-death pathways, yet how membrane organisation connects these adaptations is not fully resolved. We therefore examined whether B4GALNT1, a ganglioside biosynthetic glycosyltransferase, couples changes in ganglioside composition and lipid-raft architecture to ferroptosis regulation and caspase-1/GSDMD-associated signalling in sorafenib-resistant HCC.

METHODS

We combined TCGA-LIHC analysis, single-cell transcriptomics, pathway-level analyses, and graph attention network modelling with a treatment-annotated local cohort of 92 patients with HCC who received sorafenib. Functional experiments were conducted in Huh7/Huh7-SR cells and additional liver-tumour models using B4GALNT1 gain- and loss-of-function, an independent second-sgRNA knockout clone, GM1 rescue, and re-expression of either wild-type B4GALNT1 or the catalytic-loss D358A variant. Validation included ganglioside profiling, detergent-resistant membrane isolation, lipid-raft disruption/repletion, ferroptosis-directed pharmacology, cell-death measurements, and xenograft studies.

RESULTS

B4GALNT1 was enriched in tumour-associated hepatocyte populations and was upregulated in Huh7-SR cells. In the local cohort, higher B4GALNT1 protein abundance tracked with less favourable response to sorafenib and poorer survival outcomes. Increasing B4GALNT1 shifted ganglioside abundance toward GM2/GM1/GD2 and increased GM1-enriched raft-associated fractions, whereas knockout produced reciprocal changes. B4GALNT1-high cells showed increased GPX4 and SLC7A11, reduced lipid peroxidation and labile Fe2 +, and lower ferroptotic injury. At the same time, caspase-1/GSDMD-associated signalling and IL-1β/IL-18 release increased without widespread terminal membrane lysis. Exogenous GM1 partially rescued knockout-associated changes, and wild-type B4GALNT1 restored the phenotype more effectively than the D358A mutant. Concordant directional effects were observed in additional cell models and in xenografts.

CONCLUSIONS

The data are consistent with a model in which B4GALNT1 reshapes ganglioside composition and GM1-enriched membrane microdomains, thereby supporting a drug-adapted state that combines greater ferroptosis resistance with restricted caspase-1/GSDMD-associated membrane permeabilisation. This integrated phenotype accompanies reduced sorafenib responsiveness and offers a mechanistic basis for acquired resistance in HCC.

HIGHLIGHTS: B4GALNT1 is associated with sorafenib resistance and poorer outcomes in treated HCC. B4GALNT1 remodels gangliosides and lipid rafts, limiting ferroptotic injury. Caspase-1/GSDMD signalling increases without widespread terminal membrane lysis. Genetic rescue and cross-model studies support B4GALNT1 specificity.