Abstract 325: The cellular prion protein is involved in the modulation of Wnt signaling in glioblastoma

Abstract

Abstract Glioblastoma (GBM) is an aggressive, invasive and therapy resistant brain tumor. Our research group has been unraveling the role of the cellular prion protein (PrPC) on GBM biology. PrPC is a scaffold protein that interacts with a plethora of partners, and it is important for proliferation and stemness maintenance of glioblastoma stem-like cells (GSCs), that are responsible for the aggressiveness and resistance to therapy of GBM. GSCs have aberrant activation of several signaling pathways that control self-renewal and differentiation, such as the Notch, Hedgehog (Hh) and Wnt pathways. Additionally, PrPC was proposed as a modulator of the Wnt signaling pathway on proliferating intestinal epithelial cells due to its binding to beta-catenin and upregulation of the transcriptional activity of the beta-catenin/TCF7L2 complex. Therefore, we aim to further explore this connection between Wnt signaling and PrPC in GBM. We generated U87 and U251 GBM PrPC knockout cells (PrPC-KO) by CRISPR-Cas9. RNA sequencing analysis comparing PrPC-KO GBM cells with wild-type controls resulted in 28 differentially expressed genes (DEGs) for U87 cells and 10 DEGs for U251 cells that are components of pathways related to Wnt signaling. We chose to analyze the expression of the upregulated DEGs CAMK2A, NFATC4, WISP1, PPP2R2B, SFRP2; and the downregulated DEGs RAC3 and SHH by qPCR. Our results indicate that the Wnt-Ca2+ pathway may be activated in U87 PrPC -KO cells, due to increased expression of NFATC4 and CAMK2A. Regarding the canonical Wnt signaling, it appears to be a balance in the expression of genes that activate or inhibit these pathways in U87 PrPC-KO cells, since WISP1 and PPP2R2B were upregulated and SFRP2 was downregulated. When analyzing the expression of the same genes in U251 PrPC-KO cells, however, we observed a different expression profile than what was found for U87 cells. Apart from RAC3, the expression of all genes analyzed were downregulated. Importantly, expression of SHH was downregulated in both U87 and U251 PrPC-KO cells. When we analyzed protein levels of Wnt signaling components, we found a decrease of beta-catenin and Wnt5a/b in U87 PrPC-KO, demonstrating a downregulation of the canonical signaling. Moreover, we found by immunofluorescence that beta-catenin distribution on the cellular membrane was altered in U87 PrPC-KO cells. Our results demonstrate, therefore, a potential involvement of PrPC in modulating Wnt signaling in GBM. As a scaffolding protein, PrPC may be contributing to GBM biology by increasing the effectiveness of Wnt signaling, by localizing beta-catenin on important regions of the tumor cell membrane, or by insulating beta-catenin from degradation. Altogether, PrPC may be a promising target for the development of novel therapy strategies against GBM. Supported by FAPESP (2019/14952-0) Citation Format: Barbara Paranhos Coelho, Mariana B. Prado, Frederico M. Ferreira, Tiago G. dos Santos, Helder I. Nakaya, Marilene H. Lopes. The cellular prion protein is involved in the modulation of Wnt signaling in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 325.