Prion protein regulates invasiveness in glioblastoma stem cells

Abstract

Background: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP^C), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP^C expression on GBM.

Methods: To address this goal, we employed CRISPR-Cas9 technology to generate PrP^C knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP^C-wild-type (WT) cells and PrP^C-knockout (KO) cells were used to identify pathways regulated by PrP^C. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP^C in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.

Results: Transcriptome analysis of PrP^C-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP^C-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP^C disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP^C levels through CD44 overexpression further emphasizes their regulatory role in these processes.

Conclusions: These findings establish PrP^C as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.

Keywords: CD44; Cellular prion protein; Glioblastoma stem cells; Invasion; Migration.

Fig. 1

Characterization of U87 and U251 PrP^C KO cells. (a) Illustration of the study design for the generation of PrP^C KO cells. (b) RT-qPCR of PRNP mRNA amplifying the region inside the deletion site (inDEL) in U87 cells (n = 4; **P < 0.01; ****P < 0.0001). (c) Expression of PrP^C protein in U87 and U251 WT and KO cells, in monolayer (M) and neurosphere (N) conditions (left) and analysis of the expression of PrP^C in WT cells through band densitometry (right). Ratio between PrP^C and Actin (n = 3; *P < 0.05; ***P < 0.001; ****P < 0.0001). (d) Histogram of cell surface expression of PrP^C in U87 WT and KO neurosphere cells, and in U251 WT and KO monolayer cells. (e) Heatmaps depicting the relative gene expression of stem cell markers in U87 and U251 KO monolayer cells and WT and KO neurosphere cells, in relation to their monolayer WT counterparts. Asterisks (*) represent a comparison with the monolayer WT group, and plus signs (+) represent a comparison with the neurosphere WT group (p values are described in the Results section). (f) Histograms of cell surface expression of CD133 and SSEA1 proteins in U87 WT and PrP^C KO neurospheres. (g) Growth curve of U87 and U251 WT and KO cells in monolayer condition (n = 6) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 comparing WT vs. KO of the same cell line; ⁺⁺⁺⁺p < 0.0001 comparing U87 WT vs. U251 WT cells). (h) Self-renewal assays measuring the number of neurospheres in U87 and U251 WT and KO cells (n=*P < 0.05; **P < 0.01)

Fig. 2

Bulk RNA-Seq data analysis shows that PrP^C may modulate migration, proliferation, and stemness-related genes. (a) Volcano plots of the comparison between U87 and U251 WT versus PrP^C -KO cells. The plots depict non-significant (gray), downregulated (blue), and upregulated (red) differentially expressed genes (DEGs). (b) Dot plot of key DEGs found in U87 and U251 cells, with size and color relating to p-value and fold change, respectively. (c) Overrepresentation analysis of U87 cells and U251 DEGs. (d) Gene expression of selected DEGs related to stemness maintenance, cellular migration, and invasion in U87 and U251 KO monolayer cells, in relation to their monolayer WT counterparts (p values are described in the Results section)

Fig. 3

Loss-of-PrP^C disrupts CD44 expression and localization and decreases cell invasiveness. (a) Protein levels of CD44 in U87 and U251 WT and KO cells in monolayer and neurosphere conditions. Analysis of the expression of CD44 through band densitometry, with the ratio between CD44 and GAPDH. (b) Western blot analysis showing elevated PrP^C levels in CD44-overexpressing cells. Densitometric analysis of PrP^C and HSP90 (loading control) was performed for U87 WT and PrP^C knockout (KO) cells transfected with CD44-GFP or PrP^C-GFP (untransfected cells as control). Statistical analysis was conducted using a two-way ANOVA followed by Bonferroni’s post-hoc test (n = 3; ****P < 0.0001, ***P < 0.001). (c) Immunofluorescence of CD44 (green), PrP^C (red), and DAPI (blue) in U87 and U251 WT and KO monolayer and neurosphere conditions. Inserts in WT show co-localization of CD44 and PrP^C. Inserts panel in KO shows CD44 assemblies, Scale bar = 15 μm. (d) Pearson’s correlation coefficient was calculated to quantify colocalization between CD44 and PrP^C signals, with values presented in the graph. (e-g) Representative photomicrographs of U87 and U251WT and KO in monolayer and neurosphere conditions, for cellular migration or invasion through transwell assays, and graphical representation of the number of cells that migrated per quadrant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (h) Invasion assays confirmed previous findings, showing that PrP^C KO reduces invasiveness in GBM cells, both in untransfected (UNT) and CD44-overexpressing conditions. A general decrease in cell invasion was observed in both WT and KO cells following CD44 transfection (*P < 0.05)

Fig. 4

PRNP expression in patient tumor samples predicts lower survival and may be associated with migration and invasion pathways in glioblastoma stem cells. (a) Correlation between PRNP expression and GCS’s regulators obtained from the TCGA database. (b) Curves showing survival probabilities in patients with varying PRNP (right) or CD44 expression (left). Survival and expression data were obtained from Gliovis, using the TCGA GBM dataset. (c-e) correlation analysis between CD44 and PRNP expression in GBM subtype samples. Scatter plot illustrating the relationship between CD44 and PRNP. The line represents the best-fit linear regression, with the Pearson correlation coefficient (r) and significance level (p-value) indicated in the top left corner. (f) t-distributed stochastic neighbor embedding (UMAP) graphic showing GSCs cultivated as spheres (n = 10,536) distributed according to transcription patterns. Different colors represent each. (g) Violin plot shows PRNP expression in each cluster. Central dots represent the means. (h) Gene Set Enrichment Analysis (GSEA) of cluster 10 showing main upregulated pathways. (i-j) Enrichment score of cell migration-related pathways (i) and cell growth-related pathways (j)

Fig. 5

PrP^C knockout impairs the invasiveness of glioblastoma. PrP^C knockout (KO) in glioblastoma cells altered the expression of genes involved in cell migration, invasion, and stemness pathways. Notably, the cell lines U87 and U251 showed distinct expression patterns of these genes but had similar phenotypic responses to PrP^C-KO, i.e., decreased proliferation, self-renewal, and migration and invasion capabilities. We also found that PrP^C-KO led to a decrease in total protein levels of CD44 and of cell surface levels of SSEA1, CD44, and CD133. We propose that the lack of PrP^C leads to a disruption of the interaction between CD44 and extracellular matrix components that impair GBM cell motility