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

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. Immunofluorescent staining indicated mostly intracellular localization of PANX1 in GBM 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 β-catenin mRNA and other components of the Wnt pathway. The deletion of PANX1 resulted in a disruption of the β-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. The intracellular PANX1 staining was unaffected by channel inhibition; however, both blockers dramatically decreased F-actin filament formation. PANX1 and b-actin protein levels remained unchanged with blocker treatment, but the cellular localization of b-catenin became more intracellular compared to controls upon drug treatments. Xenografted GBM tumours grown on a chick chorioallantoic membrane model showed a reduction in tumour cell viability by bioluminescent imaging and reduced hemorrhaging incidence when treated with PBN. PBN-treated tumours had reduced pro-inflammatory IL-8 mRNA expression, a cytokine involved in vascular leakage. We provide evidence that PANX1 is upregulated in GBM, and PANX1 inhibition or deletion reduces several tumorigenic properties of GBM cells across multiple model systems. 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.