The expression of pro-prion, a transmembrane isoform of the prion protein, leads to the constitutive activation of the canonical Wnt/β-catenin pathway to sustain the stem-like phenotype of human glioblastoma cells.

Background: Cellular prion protein (PrP^C) is a widely expressed membrane-anchored glycoprotein, which has been associated with the development and progression of several types of human malignancies, controlling cancer stem cell activity. However, the different molecular mechanisms regulated by PrP^C in normal and tumor cells have not been characterized yet.

Methods: To assess the role of PrP^C in patient-derived glioblastoma stem cell (GSC)-enriched cultures, we generated cell lines in which PrP^C was either overexpressed or down-regulated and investigated, in 2D and 3D cultures, its role in cell proliferation, migration, and invasion. We evaluated the role of PrP^C in supporting GSC stemness and the intracellular signaling involved using qRT-PCR, immunocytofluorescence, and Western blot.

Results: Stable PrP^C down-regulation leads to a significant reduction of GSC proliferation, migration, and invasiveness. These effects were associated with the inhibition of the expression of stemness genes and overexpression of differentiation markers. At molecular level PrP^C down-regulation caused a significant inhibition of Wnt/β-catenin pathway, through a reduced expression of Wnt and Frizzled ligand/receptor subtypes, resulting in the inhibition of β-catenin transcriptional activity,  as demonstrated by the reduced expression of its target genes. The specificity of PrP^C in these effects was demonstrated by rescuing the phenotype and the biological activity of PrP^C down-regulated GSCs by re-expressing the protein. To get insights into the distinct mechanisms by which PrP^C regulates proliferation in GSCs, but not in normal astrocytes, we analyzed structural features of PrP^C in glioma stem cells and astrocytes using Western blot and immunofluorescence techniques. Using Pi-PLC, an enzyme that cleaves GPI anchors, we show that, in GSCs, PrP is retained within the plasma membrane in an immature Pro-PrP isoform whereas in astrocytes, it is expressed in its mature PrP^C form, anchored on the extracellular face of the plasma membrane.

Conclusions: The persistence of Pro-PrP in GSCs is an altered cellular mechanism responsible of the aberrant, constitutive activation of Wnt/β-catenin pathway, which contributes to glioblastoma malignant features. Thus, the activity of Pro-PrP may represent a targetable vulnerability in glioblastoma cells, offering a novel approach for differentiating and eradicating glioblastoma stem cells.