Novel Pannexin 1 isoform is increased in cancer

Pannexin 1 (PANX1) is upregulated in many cancers, where its channel activity and signalling promote tumorigenic properties. Here, we report that potential internal translation start sites exist in mouse and human PANX1 which have implications in trafficking and protein interaction. Using mouse PANX1 constructs for each internal methionine (M) we saw that the shorter PANX1 isoforms were glycosylated, able to traffic to the cell surface and PANX1-M37 formed channels which could be activated by C-terminus cleavage or 1-adrenoceptor stimulation. Furthermore, we report a novel [~]25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer, and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, and using Expasy PeptideCutter we did not find any evidence of hPANX1 cleavage sites which would produce a 25 kDa fragment, suggesting a potential alternative translation initiation site as the source of hPANX1-25K. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at multiple sites including the canonical N255 and novel N338 and N394 residues, and can interact with both {beta}-catenin and full length hPANX1. Using cell surface biotinylation and immunocytochemistry, we also determined hPANX1-25K exhibits a predominantly intracellular localization. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and patient-matched normal skin, indicating that it may be differentially regulated in normal and cancer cells.