Prion Protein Expression is Correlated with Glioma Grades

Dear Editor,

Glioma is the most common primary central nervous system (CNS) tumors in human. Gliomas are classified into low-grade glioma (LGG, grades I–II) and high-grade glioma (HGG, grades III–IV) according to the World Health Organization classification guidelines (Louis et al. 2007). Comparing to the LGG, the HGG becomes more aggressive and has a poorer median overall survival. At present, glioma is mainly treated by radiotherapy, chemotherapy and surgical resection, but the effect is not ideal, especially for the glioblastoma (GBM), which is the most malignant type of gliomas and is resistant to current therapy protocols. There are several theories about the pathogenesis of glioma, such as radiation (Ohgaki and Kleihues 2005), genetics (Farrell and Plotkin 2007; Goodenberger and Jenkins 2012) and viruses (Lawler. 2015; Xing et al. 2016), while its etiology remains obscure. Therefore, it is urgent to investigate the causes and to identify the promising molecules that target the glioma.

Cellular prion protein (PrP^C) is a conserved ubiquitously expressed glycoprotein most abundant in CNS, which is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) group (Baiardi et al. 2019; Sarnataro et al. 2017). The misfolding form of PrP^C is responsible for a range of neurodegenerative diseases including Creutzfeldt-Jakob, familial fatal insomnia, and kuru, etc. (Atkinson et al. 2016). Although the physiological role of PrP^C is not completely defined, several evidences propose that PrP^C has been attributed to cell adhesion, anti-apoptosis, migration, signaling, viral replication, immune modulation, and cell differentiation (Sarnataro et al. 2017). In some tumors such as oral squamous cell carcinoma, colon cancer, breast cancer, gastric cancer and melanoma, the expression level of PrP is upregulated (Yang et al. 2014). As for glioma, previous studies mainly focused on GBM and were usually performed on cell lines or animal models (Barbieri et al. 2011; Corsaro et al. 2016; Iglesia et al. 2017; Lopes et al. 2015; Zhuang et al. 2012). There are rare researches regarding the PrP expression in glioma of different grades. To explore the association between PrP and glioma progression, we detected the expression of PrP in 66 glioma tissues of different grades and 15 control brain tissues from patients with brain trauma, including cortex of occipital lobe, frontal lobe, parietal lobe and temporosphenoid lobe, by immunohistochemistry (IHC).

A total of 66 patients with glioma, who were diagnosed according to the World Health Organization classification of brain tumors, underwent surgical resection from January 2007 to September 2013 in Beijing Tiantan Hospital were enrolled in this study. Among the 66 glioma cases, there were 34 (51.5%) HGG and 32 (48.5%) LGG. The patients included 39 (59.1%) men and 27 (40.9%) women, with an average of 43.4 years old (range 12–78 years). The male:female ratio in the enrolled patients was 1.4. Fifteen control brain tissues were obtained from brain trauma surgery.

The antibody against PrP was produced by immunization with immunogen of the synthetic peptide within Human Prion protein PrP aa 200–300 (C terminal) and purchased from Abcam, America (ab52604). The IHC was performed according to our previous research (Xing et al. 2016). Briefly, the tissues were incubated overnight at 4 C with 1:800 dilution of a primary antibody against PrP, followed by a secondary horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody (PV9001, Zhongshan Golden Bridge Bio Co., Ltd., China). Finally, the 3, 30-diaminobenzidine (DAB) was added as a chromogen. After counterstaining with hematoxylin, the staining was evaluated. The negative controls were performed using phosphate buffer instead of the primary antibody.

The immunostaining results were evaluated by a standard scoring methodology. Five high-power fields were randomly selected from each section, and the positive expression intensity and distribution range of the positive cells in the sections were scored. The staining intensity was scored as 0 (colorless), 1 (light yellow), 2 (yellow or brown), and 3 (dark brown); the percentages of positive cells were denoted as 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%). The multiplication of both scores was used to evaluate the immunostaining results, as follows: overall scores of B 3, ≤ 3–6, and > 6 were defined as negative, weak positive, and strong positive, respectively.

Statistical analysis was performed using SPSS 22.0 (SPSS Inc.) The Mann–Whitney U test were used to determine the differences between the PrP expression in glioma of different grades. The Mantel–Haenszel Chi square test was used to determine whether there was a linear relationship between the positive degree of PrP staining and glioma grades. The number of positive degree of PrP staining was 1–3, and the grade of glioma was 1–4. Of the statistical analysis, P < 0.05 was considered statistically significant.

Of the 66 glioma specimens, 24 cases (36.4%) showed positive immunoreactivity for PrP, which was mainly detected in cytoplasm and nucleus of tumor cells, while positive staining was observed only in 1 case of control brain tissues with detection rate 6.7% (1 of 15) (Fig. 1A and Table 1). Of note, 47.0% (16 of 34) of the HGG and 25.0% (8 of 32) of the LGG tissues showed positive staining. Moreover, strong positive staining of PrP was observed in 29.4% (10 of 34) of the HGG tissues, but only in 6.3% (2 of 32) of the LGG tissues. In the semiquantitative analysis, there was a significant difference in the positive rate and expression level of PrP between the HGG and LGG (P < 0.05) (Table 1). Notably, the results of Mantel–Haenszel Chi square test showed a linear relationship between the positive degree of PrP staining and glioma grades, χ² = 6.187, P = 0.013. Pearson correlation results showed that R = 0.309, P = 0.012 (Fig. 1B). These results suggest that the PrP expression is in association with glioma grades and may play a role in glioma progression.

Figure 1.  A PrP expression in control brain tissues and glioma tissues of different grades detected by IHC. Representative images were shown at low or high magnification. Bar = 100 lm. B Scatterplot of positive degree of PrP staining on the glioma of different grades. χ ² = 6.187, P = 0.013. Pearson correlation results showed that R = 0.309, P = 0.012

The mature form of PrP^C is a widely expressed, highly conserved GPI anchored, cell-surface glycoprotein, which can bind to more than 50 ligands and therefore participate in a plenty of biological responses, such as apoptosis, cell adhesion, migration, proliferation, pro-inflammatory cytokine production, metal homeostasis, signal transduction, and regulation of transcription (Hirsch et al. 2017). As the most malignant glioma, GBM contains a subpopulation of glioblastoma stem-like cells (GSCs) that participate in tumor maintenance, invasion, and therapeutic resistance (Lathia et al. 2015). Therefore, GSCs could be a promising target for treatment of GBM. Previous researches have investigated the role of PrP^C in GSCs. For instance, it has been proved that PrP^C expression is directly correlated with the proliferation rate of the CSC cells. CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrP^C down-regulated cells. Moreover, PrP^C down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression) (Corsaro et al. 2016). Additionally, PrP^C and its partner, co-chaperone Hsp70/90 organizing protein (HOP, aka stress-inducible phosphoprotein 1, STI1 or STIP1) are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grade I–III (Lopes et al. 2015). Furthermore, PrP^C and HOP also play a role in GBM tumorigenesis and in neural stem cell maintenance, therefore high levels of PrP^C and HOP are associated with greater GBM proliferation and lower patient survival. The modulation of HOP–PrP^C engagement or the decrease of PrP^C and HOP expression may represent a potential therapeutic intervention in GBM, regulating GSC cell self-renewal, proliferation, and migration (Iglesia et al. 2017). Besides the relationship between PrP^C and CSCs of GBM, PrP^C also participates in the progression of glioma through promoting the survival (Zhuang et al. 2012), proliferation and migration (Fonseca et al. 2012; Erlich et al. 2007) of human glioma cells and protecting glioma cells from autophagy-dependent cell death (Barbieri et al. 2011) by binding to different partners. Moreover, depleting PrP^C inhibits growth, promotes programmed cell death in gliomas (Barbieri et al. 2011). Regarding to the relationship between PrP^C and GSCs, the upregulation of PrP may highly indicate the glioma progression.

In conclusion, our study investigated the PrP expression in gliomas of different grades, the results showed that the PrP expression is correlated with glioma grades, and PrP staining degree is in linear relationship with the glioma grades, indicating that PrP may play an important role in the glioma progression, and could be a promising biomarker for the prognosis of glioma.

This work was supported by the following funds: the National Natural Science Foundation of China (81671971, 81871641 and 81701992) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201810025035).