Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/β-catenin pathway, because β-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

Pannexins (Panxs), large-pore channel forming glycoproteins, are expressed in a wide variety of tissues including the skin, bone, and cochlea. To date, the use of single knockout mouse models of both Panx1 and Panx3 have demonstrated their roles in skin development, bone formation, and auditory phenotypes. Due to sequence homology between Panx1 and Panx3, when one Panx is ablated from germline, the other may be upregulated in a compensatory mechanism to maintain tissue homeostasis and function. To evaluate the roles of Panx1 and Panx3 in the skin, bone, and cochlea, we created the first Panx1/Panx3 double knockout mouse model (dKO). These mice had smaller litters and reduced body weight compared to wildtype controls. The dKO dorsal skin had decreased epidermal and dermal area as well as decreased hypodermal area in neonatal but not in older mice. In addition, mouse skull shape and size were altered, and long bone length was decreased in neonatal dKO mice. Finally, auditory tests revealed that dKO mice did not exhibit hearing loss and were even slightly protected against noise-induced hearing damage at mid-frequency regions. Taken together, our findings suggest that Panx1 and Panx3 are important at early stages of development in the skin and bone but may be redundant in the auditory system. Key messages & Panx double KO mice had smaller litters and reduced body weight. & dKO skin had decreased epidermal and dermal area in neonatal mice. & Skull shape and size changed plus long bone length decreased in neonatal dKO mice. & dKO had no hearing loss and were slightly protected against noise-induced damage.

Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains, using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H), is present in cancer cells, but was not different from the wildtype (Q5) in glycosylation, trafficking, or channel function, and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1, with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumour, prevented phosphorylation at Y150 as well as complex N-glycosylation, while increasing intracellular localization. SRC is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating post-translational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.

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