Prado Mariana B, Coelho Bárbara P, Iglesia Rebeca P, Alves Rodrigo N, Boccacino Jacqueline M, Fernandes Camila F L, Melo-Escobar Maria Isabel, Ayyadhury Shamini, Cruz Mario C, Santos Tiago G, Beraldo Flávio H, Fan Jue, Ferreira Frederico M, Nakaya Helder I, Prado Marco A M, Prado Vania F, Duennwald Martin L, Lopes Marilene H
Laboratory of Neurobiology and Stem Cells, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
BMC Cancer. 2024 Dec 18;24(1):1539. doi: 10.1186/s12885-024-13285-4.
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), 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 expression on GBM.
To address this goal, we employed CRISPR-Cas9 technology to generate PrP 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-wild-type (WT) cells and PrP-knockout (KO) cells were used to identify pathways regulated by PrP. 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 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.
Transcriptome analysis of PrP-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-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP levels through CD44 overexpression further emphasizes their regulatory role in these processes.
These findings establish PrP as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.
胶质母细胞瘤（GBM）是一种由胶质母细胞瘤干细胞（GSCs）驱动的侵袭性脑肿瘤，GSCs是治疗干预的一个有吸引力的靶点。细胞朊蛋白（PrP）是一种参与多种细胞过程的支架蛋白，它与各种膜和细胞外基质分子相互作用，影响肿瘤生物学。在此，我们研究PrP表达对GBM的影响。
为实现这一目标，我们采用CRISPR-Cas9技术生成PrP敲除（KO）胶质母细胞瘤细胞系，以进行详细的功能丧失研究。对U87或U251 PrP野生型（WT）细胞和PrP敲除（KO）细胞进行批量RNA测序，随后进行差异表达基因和通路富集分析，以确定受PrP调控的通路。免疫荧光分析用于评估细胞形态和蛋白质分布。为评估蛋白质水平，采用了蛋白质印迹法和流式细胞术分析。Transwell分析和生长曲线分析分别用于确定PrP缺失对GBM侵袭性和增殖的影响。利用来自癌症基因组图谱（TCGA）和布罗德研究所单细胞数据门户的患者肿瘤单细胞RNA测序数据分析，评估我们的体外结果与患者样本之间的相关性。
对PrP-KO GBM细胞系的转录组分析显示，与关键肿瘤进展通路相关的基因表达发生改变，包括迁移、增殖和干性。这些发现得到了实验的证实，实验显示PrP-KO GBM细胞的侵袭、迁移、增殖和自我更新受损，突出了其在维持肿瘤生长中的关键作用。值得注意的是，PrP缺失破坏了关键干性标志物的表达和定位，尤其是CD44。此外，通过CD44过表达调节PrP水平进一步强调了它们在这些过程中的调节作用。
这些发现确立了PrP作为GSCs细胞表面重要分子的调节剂，突出了其作为GBM治疗靶点的潜力。
