EREG is the core onco-immunological biomarker of cuproptosis and mediates the cross-talk between VEGF and CD99 signaling in glioblastoma

Background: Glioma is the most prevalent primary tumor of the central nervous system. Glioblastoma multiforme (GBM) is the most malignant form of glioma with an extremely poor prognosis. A novel, regulated cell death form of copper-induced cell death called "cuproptosis" provides a new prospect for Cancer treatment by regulating Cuproptosis.

Methods: Data from bulk RNA Sequencing (RNA-seq) analysis (The Cancer Genome Atlas cohort and Chinese Glioma Genome Atlas cohort) and single cell RNA-seq (scRNA-seq) analysis were integrated to reveal their relationships. A scoring system was constructed according to the cuproptosis-related gene expression, and core genes were experimentally verified using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF). Moreover, cell counting kit-8 (CCK8), colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, transwell, and flow cytometry cell cycle were performed to evaluate cell proliferation, invasion, and migration.

Results: The Cuproptosis Activation Scoring (CuAS) Model has stable and independent prognostic efficacy, as verified by two CGGA datasets. Epiregulin (EREG), the gene of the model has the most contributions in the principal component analysis (PCA), is an onco-immunological gene that can affect immunity by influencing the expression of programmed death-ligand 1 (PD-L1) and mediate the process of Cuproptosis by influencing the expression of ferredoxin 1 (FDX1). Single cell transcriptome analysis revealed that high CuAS GBM cells are found in vascular endothelial growth factor A (VEGFA) + malignant cells. Oligodendrocyte transcription factor 1 (OLIG1) + malignant is the original clone, and VEGF and CD99 are the differential pathways of specific cell communication between the high and low CuAS groups. This was also demonstrated by immunofluorescence in the tissue sections. Furthermore, CuAS has therapeutic potential for immunotherapy, and we predict that many drugs (methotrexate, NU7441, KU -0063794, GDC-0941, cabozantinib, and NVP-BEZ235) may be used in patients with high CuAS.

Conclusion: EREG is the core onco-immunological biomarker of CuAS and modulates the cross-talk between VEGF and CD99 signaling in glioblastoma, and CuAS may provide support for immunotherapy and chemotherapy.

Bulk RNA-seq analysis; CD99; Cuproptosis; Flow cytometry cell cycle; Immune microenvironment; Immunohistochemistry; Single cell RNA-seq; Transwell assays; VEGFA.