123 Ubiquitination control of tumor proliferation, metastasis and immune escape via prohibitin/stat3/PD-L1 axis in melanoma

Ubiquitin editing enzyme TNFAIP3 (also called A20) is well known for its immunosuppressive functions. However, few studies has investigated functions on tumor intrinsic A20. Here, we report A20 promotes melanoma growth, metastasis, and immune evasion through prohibitin/Stat3/PD-L1 axis. Tissue microarray, GO database analysis, western blot and qRT-PCR showed the elevation of melanoma A20 expression in vivo and in vitro. Additionally, A20 expresses higher in metastasis melanoma compared with primary melanoma, both in xenograft mice and melanoma patients. Xenograft assays, western blot and flow cytometry revealed A20 significantly promoted melanoma proliferation both in vitro and in vivo. Cell immunofluorescence, mouse tail intravenous injection and transwell assays demonstrated A20 facilitated melanoma metastasis. Flow cytometry and TCGA database analysis uncovered A20 assisted melanoma immune evasion through increasing PD-L1 expression. ChIP and co-IP assays confirmed A20 transcriptionally regulated PD-L1 expression by activating prohibitin-Stat3 pathway. Through co-IP assay, we confirmed that A20 linked ubiquitin chain on prohibitin, which degraded prohibitin and subsequently promoted stat3 phosphorylation on Y705 subsite. In conclusion, tumor intrinsic A20 promotes melanoma growth, metastasis, and immune evasion via STAT3/PD-L1 axis.