RNF182 induces p65 ubiquitination to affect PDL1 transcription and suppress immune evasion in lung adenocarcinoma

Abstract

Background The RING finger (RNF) proteins are a large group of ubiquitin ligases whose aberrant expression is often associated with disease progression. This study examines the function of RNF protein 182 (RNF182) in lung adenocarcinoma (LUAD) cells and its impact on p65 and programmed death ligand 1 (PDL1) regulation. Methods Expression of RNF182, p65, and PDL1 in LUAD tissues and cells was measured using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT‐qPCR), and/or western blot (WB) assays. LUAD cells were induced to overexpress RNF182 and p65, followed by cell counting kit‐8, colony formation, Transwell, and flow cytometry assays to evaluate the cells’ malignant phenotype. Coimmunoprecipitation and WB assays were used to verify RNF182's effect on p65 ubiquitination. Chromatin immunoprecipitation‐qPCR and luciferase assays were used to analyze p65's transcriptional regulation of PDL1. Coculture of LUAD with CD8+ cytotoxic T cells was performed to detect lactate dehydrogenase release and interferon‐γ and interleukin‐2 concentrations. LUAD cells were implanted in mice to analyze tumorigenicity. Results RNF182 was poorly expressed, while p65 and PDL1 were highly expressed in LUAD tissues and cells. RNF182 overexpression suppressed the malignant properties of LUAD cells, and it promoted p65 ubiquitination and protein degradation. p65 activated PDL1 transcription. Overexpression of RNF182 suppressed the PDL1 expression, increased the cytotoxicity in LUAD cells cocultured with CD8+ T cells, and suppressed the tumorigenesis of cancer cells in vivo. However, these tumor‐suppressive effects of RNF182 on LUAD cells were blocked by p65 restoration. Conclusion This research demonstrates that RNF182 induces p65 ubiquitination to suppress PDL1 transcription and immunosuppression in LUAD.

Post-translational modifications of p65: state of the art

“…Through regulating protein ubiquitination, the E3 participateS in various cellular physiological processes, underscoring its importance in the ubiquitin pathway. Numerous NF-κB ubiquitination-related regulatory factors have been identified, including E3 ligases such as SOCS1 ( Maine et al, 2007 ), PPARγ ( Hou et al, 2012 ), ING4 ( Hou et al, 2014 ), RNF182 ( Zeng et al, 2023 ), ORF73 ( Rodrigues et al, 2009 ), among others.…”

“…ING4 not only functions in tumorigenesis but also plays a critical role in the negative regulation of inflammation ( Hou et al, 2014 ). RNF182, identified as a novel tumour-suppressive E3 ubiquitin ligase, induces p65 ubiquitination, suppressing PDL1 transcription and immune suppression in lung adenocarcinoma, thus mitigating cancer progression ( Zeng et al, 2023 ). PPARγ, a peroxisome proliferator-activated receptor, acts as an E3 ligase facilitating p65 ubiquitination and degradation through its RING domain.…”

Role of Ubiquitination and Epigenetics in the Regulation of AhR Signaling in Carcinogenesis and Metastasis: “Albatross around the Neck” or “Blessing in Disguise”

“…RNF182 (Ring Finger Protein-182) is an E3 ubiquitin ligase [77,78]. RNF182 knockdown considerably increased colony formation and proliferation of the cancer cells.…”