A direct Pannexin1 is a potential target for the treatment of malignant melanoma

Introduction: Melanoma is the most aggressive type of skin malignancy with increasing incidence worldwide. Previously, we have shown that the channel forming protein, Pannexin1 (PANX1), regulates several cellular processes in melanoma cells including proliferation, migration and invasion/metastasis in vitro and ex vivo. However, the mechanism through which PANX1 mediates melanoma progression has not been elucidated. We have observed that knocking down PANX1 decreases the abundance of -catenin, a key transcription factor in the Wnt signaling pathway. Wnt pathway regulates several processes in melanoma cells, including proliferation, migration and metastasis. These observations prompted us to evaluate the potential crosstalk between PANX1 and components of the Wnt signaling pathway.Hypothesis: We hypothesized that PANX1 modulates melanoma progression through crosstalk with the Wnt signaling pathway.Materials and Methods: In silico analysis was conducted to determine whether there is any correlation between PANX1 and -catenin mRNA in samples from patients with malignant melanoma or breast carcinoma. We used immunoprecipitation studies, as well as in vitro analysis using pure proteins to evaluate the interaction of PANX1 with -catenin, a key transcription factor in the Wnt signaling pathway. We used CRISPR/Cas9 system to generate PANX1 knock out cells. PANX1 and -catenin knockdown cells were generated using shRNA and further used in the functional assays, such as proliferation and mitochondrial metabolic stress test. We used PANX1 blockers, Probenecid (PBN) and Spironolactone (SPL), to inhibit PANX1 channel function and determine their impact on the abundance and subcellular localization of -catenin. Confocal microscopy was used to evaluate the subcellular localization of -catenin in PANX1-deficient melanoma cells. We used RT-PCR to determine changes in the mRNA levels of Wnt pathway effector proteins, such as -catenin, MITF and LEF1 in PANX1-deficient cells.Results: In this study, we demonstrated that PANX1 binds to -catenin and modulates its subcellular localization. PANX1 and -catenin co-immunoprecipitated from several established melanoma cell lines. The interaction of PANX1 -catenin and has a functional impact on melanoma cells. In vitro analysis using pure proteins demonstrated a direct interaction between PANX1 and -catenin. Analysis with fragments of PANX1 revealed that the association is mediated through the C-terminal region of PANX1. The abundance of -catenin is significantly decreased when PANX1 is either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Fluorescence imaging showed a disrupted pattern of -catenin localization at the cell membrane in PANX1-deficient cells. Several Wnt target genes, including Microphthalmia-Associated Transcription Factor (MITF), were significantly suppressed in PANX1-deficient cells. Both PANX1- and -catenin-deficient melanoma cells grew significantly less than controls in culture. In addition, mitochondrial metabolism of PANX1-deficient cells was significantly impaired, indicating a role for PANX1 in the regulation of melanoma cell metabolic profile.Discussion and Conclusions: Here we report a previously undescribed role for PANX1 in regulation of the Wnt signaling pathway through direct interaction with -catenin, a significant effector of the Wnt signaling. The findings of our study provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and -catenin interact as a potential new component in the Wnt signaling pathway.