Hebb, M. | bioRxiv Science

bioRxiv2026

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. 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.

cancer biology↗

bioRxiv2024

Novel Pannexin 1 isoform is increased in cancer

Alternative translation initiation (ATI) is a process of increasing protein diversity from one transcript, allowing cells to rapidly respond to signals, which is particularly important in cancer cells. Here, we report potential internal translation start sites exist in PANX1 which have implications in trafficking and channel function. Using mouse (mPANX1) constructs for each internal methionine, we saw that these PANX1 isoforms were N-glycosylated, could traffic to the cell surface and mPANX1-M37 formed functional channels activated by C-terminus cleavage or 1-adrenoceptor stimulation. We also identified a ~25 kDa isoform of mPANX1 (mPANX1-25K) endogenously expressed in mouse melanoma cell lines that could be confirmed with a cognate peptide. mPANX1-25K lacks the mPANX1 N-terminus and most likely corresponds to the M210 internal translation start site since we could not identify any alternative transcripts that would produce this ATI product. When we expressed the human equivalent of M210 in Hs578T PANX1 KO cells with and without wildtype human PANX1, we determined M211 exhibits a predominantly intracellular localization, is N-glycosylated and can interact with full-length human PANX1. Collectively, these findings indicate species specific differences in the abundance of PANX1 ATI isoforms which could act independently or in conjunction with the canonical full-length protein in melanoma.

cell biology↗