Fig 2: Relationship between PRNP expression and immune infiltrating cells in BRCA.(A) Twenty-four types of immune-infiltrating cells related to PRNP expression. The absolute values of Spearman R-value are indicated by the size of round dots. (B) Scatter plots show the correlation between PRNP expression and macrophages, Th1cells, neutrophils, and B cells. (C) The heat map was drawn based on TIMER2.0 data to further evaluate the pan-cancer data for macrophages and PRNP expression was found to be correlated with macrophages M0 and M1. (D) PRNP expression was positively linked with stromal cells in BRCA. Single-cell sequencing analysis for PRNA expression in BRCA. PRNP was positively related to macrophages (E-J) and M1 macrophages (K-L).

Fig 3: PRNP expression in breast cancer tissues.Compared with normal breast tissue, a significantly reduced PRNP expression was found in (A) GSE21422, (B) GSE31192, (C) TCGA-BRCA, and (D) GEPTA2. (E) The verification of down-regulated expression of PRNP using the TNM-plot database. (F) Immunohistochemical analysis indicates downregulated PRNP levels in BRCA patients. (G) Co-expression network and enrichment pathway analysis of PRNP. Ten experimentally identified PRNP-binding molecules were acquired using STRING. (H) The top six PRNP-correlated genes were analyzed by GEPIA2, including MAP7D1, EXOC66, NCS1, PPP2R2C, ARHGEF4, and SLC9A6. (I) Heatmap representation of the prognostic relevance of the top six PRNP-correlated genes in pan-cancer. (J-K) Bar chart of GO and KEGG enrichment analysis results for PRNP in BRCA.

Fig 4: PRNP overexpression sensitizes BRCA cells to gefitinib and improves cellular ROS levels.(A) BT-549 and MDA-MB-231 cells were transiently transfected with Flag-PRNP. PRNP protein expression was measured by western blotting. (B–C) CCK8 experiments were performed and overexpression of PRNP was found to increase the inhibitory effect of gefitinib and the sensitivity of BRCA cells to gefitinib. (D–F) Overexpression of PRNP resulted in increased ROS production after gefitinib treatment. (G-I) Ferroptosis selective inhibitor ferrostatin-1 attenuated enhanced ROS production due to PRNP overexpression. (*p < 0.05, **p < 0.01).

Fig 5: Co-differentially expressed copper homeostasis genes in two BRCA datasets.(A) Three upregulated copper homeostasis genes (CASP3, SCO2, ATOX1) in BRCA datasets. (B) A downregulated copper homeostasis gene (PRNP) in BRCA datasets. (C-E) Prognostic values of the levels of expression of PRNP, CASP3, ATOX1, and SCO2 in BRCA patients indicated by Kaplan-Meier plotter database analysis.