EPHRINA5–EPH SIGNALLING MODULATES GLIOBLASTOMA MIGRATION AND PROLIFERATION VIA TRANSCRIPTIONAL REGULATION OF ADHESION GENES

Glioblastoma (GBM) is a highly invasive brain tumour in which microenvironmental cues critically influence cell motility and growth. EphrinA5, a membrane‑bound ligand for EPH receptors, has been implicated as a tumour‑suppressive signal in brain cancers, but the underlying transcriptional mechanisms remain incompletely understood. Here, we investigated how ephrinA5–EPH signalling modulates migration, proliferation and gene expression in human GBM models. U251 MG glioblastoma cells were stimulated with clustered ephrinA5‑Fc or Fc control and analysed by live‑cell imaging, EdU incorporation assays, RNA‑sequencing and RT‑qPCR. In parallel, we examined primary GBM explants cultured on Matrigel following ephrinA5‑Fc or control treatment. EphrinA5‑Fc bound robustly to the surface of U251 MG cells and significantly reduced single‑cell migratory speed over 24 h compared with Fc controls. Consistently, ephrinA5‑Fc treatment decreased the fraction of EdU‑positive U251 MG cells, indicating reduced proliferative activity. RNA‑seq of U251 MG cells revealed broad ephrinA5‑dependent transcriptional changes, including regulation of transcription factors such as ZEB1, LHX6 and NKX2.1 and of adhesion and guidance‑related genes, notably SEMA3C, DSCAML1 and NCAM1. RT‑qPCR confirmed increased ZEB1, SEMA3C and NCAM1 expression and decreased LHX6, NKX2.1 and DSCAML1 expression after ephrinA5 stimulation. Together, these data demonstrate that ephrinA5–EPH signalling constrains GBM migration and proliferation and reprograms a defined transcriptional module involving adhesion and guidance genes, providing a mechanistic entry point to dissect upstream receptor usage and downstream epigenetic regulation in glioblastoma.