Glioblastoma (GBM) is the most aggressive type of primary brain tumor, and its invasive properties contribute significantly to incomplete surgical resection and tumor recurrence. Despite extensive clinical efforts to mitigate GBM invasion, targeted therapies against GBM stem cells (GSCs), which drive invasion, remain incompletely understood. Here, we report that MGAT4A, a key glycosyltransferase involved in N-glycosylation, orchestrates EGFR-ERK1/2 signaling to promote GSC invasion. Elevated MGAT4A expression correlates with poor survival outcomes in GBM patients, highlighting its potential as a therapeutic target. Silencing MGAT4A inhibits GSC invasion and tumorigenic capacities, including self-renewal and proliferation, both in vitro and in an orthotopic brain tumor xenograft model. Proteomic N-glycosylation mapping and co-immunoprecipitation assays reveal that MGAT4A interacts with EGFR and catalyzes N-glycosylation at the EGFR N604 site. This modification triggers ERK1/2 phosphorylation, thereby driving the invasion and tumorigenic potential of GSCs. Notably, tunicamycin, an N-glycosylation inhibitor, significantly impedes GSC invasion and disrupts downstream EGFR-ERK1/2 signaling, extending survival in mice bearing orthotopic tumors. To specifically target MGAT4A, we developed a CRISPR-Cas9 sgRNA-based nanotechnology, which notably reduced GSC invasion and improved survival in mice. Collectively, our study elucidates a novel mechanistic pathway driving GSC invasion and positions MGAT4A as a promising therapeutic target for GBM.
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