Pannexin 1 inhibition reduces tumorigenic properties of patient-derived glioblastoma cells through the HIPPO and Wnt signalling pathways

Abstract

Glioblastoma (GBM) is the most common primary brain tumour, with a median survival of 12-18 months, highlighting a need for new treatment targets. We observed that pannexin 1 (PANX1), a channel-forming glycoprotein important in purinergic signalling, is upregulated in GBM compared to normal tissue and expressed throughout patient tumours. Western blot analysis of patient-derived GBM cell lines revealed significantly increased PANX1 expression in these primary lines compared to brain tissue and control glial cells. Bulk RNA-sequencing compared the gene expression of GBM cells devoid of PANX1 via CRISPR/Cas9 deletion (PANX1-KO) compared to controls. Gene Ontology and KEGG gene set analyses revealed PANX1-KO in GBM cells affects cell surface and cell junction components, processes, and pathways, including the HIPPO pathway, in addition to critically downregulating {beta}-catenin mRNA and other components of the Wnt pathway. The deletion of PANX1 resulted in a disruption of the {beta}-catenin protein and a dramatic reduction in migration and cell growth. Pharmacological inhibition of PANX1 in GBM cells with Probenecid (PBN) and Spironolactone (SPIR) demonstrated a significant reduction in live cell numbers and migration via scratch assay. Both blockers dramatically decreased F-actin filament formation, and the cellular localization of beta-catenin became more intracellular compared to controls. Xenografted GBM tumours showed a reduction in tumour cell viability by bioluminescent imaging and reduced hemorrhaging incidence when treated with PBN. These new insights support further investigation of PANX1 as a potential GBM therapeutic target and its role in multiple cancer signaling pathways that regulate this devastating disease.