EGFR N-Glycosylation Catalyzed by NDST2 Promotes Lenvatinib Resistance in Hepatocellular Carcinoma

The clinical efficacy of lenvatinib, a multitarget tyrosine kinase inhibitor used as a first-line treatment for advanced hepatocellular carcinoma (HCC), is frequently compromised by the development of drug resistance. Elucidating the molecular mechanisms underlying this resistance is essential to improving therapeutic outcomes. Using patient-derived organoids (PDO) and orthotopic HCC xenograft models, we uncovered a role for epidermal growth factor receptor (EGFR) in lenvatinib resistance. Most PDOs recapitulated the limited clinical response to lenvatinib and displayed significant resistance. Notably, resistant organoids exhibited enhanced N-glycosylation of EGFR, which correlated with increased EGFR protein expression. Functional studies demonstrated that inhibiting either global N-glycosylation or EGFR signaling restored lenvatinib sensitivity in cellular and in vivo models. Integrated proteomic and N-glycoproteomic analyses identified bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 2 (NDST2) as the key enzyme mediating site-specific N-glycosylation of EGFR at four conserved asparagine residues (N175, N196, N413, and N623). NDST2-catalyzed glycosylation enhanced EGFR stability by suppressing ubiquitin-proteasomal degradation and promoted its membrane localization, thereby activating multiple prosurvival pathways, including MAPK, PI3K/AKT, and JAK/STAT. Clinically, NDST2 was upregulated in lenvatinib-resistant HCC specimens and positively correlated with EGFR expression. Importantly, targeting NDST2 via genetic ablation or inhibition using a CaCO3 nanoparticle-based small interfering RNA delivery system effectively reversed lenvatinib resistance in HCC tumor models. These findings establish NDST2-driven EGFR N-glycosylation as a critical mechanism of lenvatinib resistance in HCC and highlight NDST2 as a promising therapeutic target for restoring drug sensitivity. SIGNIFICANCE: NDST2 catalyzes glycosylation that stabilizes EGFR and enhances downstream oncogenic signaling in hepatocellular carcinoma to confer resistance to lenvatinib, which can be overcome by targeting the NDST2-EGFR axis.