Targeting the PrPC–CD44 axis: insights into protein trafficking in glioblastoma stem-like Cells

Glioblastoma (GBM) is the most aggressive glioma subtype, with an overall survival of 1–2 years. Its remarkable cellular and molecular heterogeneity is sustained by glioblastoma stem-like cells (GSCs), a subpopulation that drives tumor maintenance, proliferation, and angiogenesis. Understanding the proteins that orchestrate GSC biology, particularly those involved in intracellular trafficking, is critical for identifying therapeutic targets. Among these players, the cellular prion protein (PrP^C), a GPI-anchored scaffold glycoprotein enriched in lipid rafts, plays a pivotal role in signaling organization. One of its ligands is CD44, a stemness tumoral biomarker highly expressed in GSCs associated with processes such as cell adhesion, migration and tumor invasion. We investigated the interaction between PrP^C and CD44 using dot blot and co-immunoprecipitation assays, confirming a direct physical interaction. In addition, immunofluorescence, qPCR, and Western blot assays revealed altered CD44 expression and localization in PrP^C knockout (KO) models. Copper-induced internalization and proteolysis inhibition assays further elucidated our understanding of their trafficking and stability. Lastly, proteomic analysis supported our experimental findings. Our results reveal a previously unrecognized role for PrP^C in regulating CD44 fate, supported by: (1) physical interaction between these two proteins; (2) reciprocal expression modulation; (3) PrP^C-dependent regulation of endocytic biomarkers; and (4) intracellular CD44 accumulation in PrP^C KO cells, indicating impaired recycling or degradation. Altogether, these findings highlight PrP^C as a key regulator of CD44 fate in GSCs and uncover a novel molecular axis in GBM pathogenesis. Targeting this interaction may represent a promising strategy to overcome therapy resistance and prevent tumor recurrence.