An in-depth understanding of immune evasion mechanisms in tumors is crucial to overcome resistance and enable innovative advances in immunotherapy. Circular RNAs (circRNAs) have been implicated in cancer progression. However, much remains unknown regarding whether circRNAs impact immune escape in non-small-cell lung carcinoma (NSCLC). We performed bioinformatics analysis to profile and identify the circRNAs mediating immune evasion in NSCLC. A luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pulldown assays and fluorescence in situ hybridization were performed to identify the interactions among circIGF2BP3, miR-328-3p, miR-3173-5p and plakophilin 3 (PKP3). In vitro T cell-mediated killing assays and in vivo syngeneic mouse models were used to investigate the functional roles of circIGF2BP3 and its downstream target PKP3 in antitumor immunity in NSCLC. The molecular mechanism of PKP3-induced PD-L1 upregulation was explored by immunoprecipitation, RIP, and ubiquitination assays. We demonstrated that circIGF2BP3 (hsa_circ_0079587) expression was increased in NSCLC and negatively correlated with CD8+ T cell infiltration. Functionally, elevated circIGF2BP3 inactivated cocultured T cells in vitro and compromised antitumor immunity in an immunocompetent mouse model, and this effect was dependent on CD8+ T cells. Mechanistically, METTL3 mediates the N6-methyladenosine (m6A) modification of circIGF2BP3 and promotes its circularization in a manner dependent on the m6A reader protein YTHDC1. circIGF2BP3 competitively upregulates PKP3 expression by sponging miR-328-3p and miR-3173-5p to compromise the cancer immune response. Furthermore, PKP3 engages with the RNA-binding protein FXR1 to stabilize OTUB1 mRNA, and OTUB1 elevates PD-L1 abundance by facilitating its deubiquitination. Tumor PD-L1 deletion completely blocked the impact of the circIGF2BP3/PKP3 axis on the CD8+ T cell response. The inhibition of circIGF2BP3/PKP3 enhanced the treatment efficacy of anti-PD-1 therapy in a Lewis lung carcinoma mouse model. Collectively, the PKP3/PD-L1 signature and the infiltrating CD8+ T cell status stratified NSCLC patients into different risk groups. Our results reveal the function of circIGF2BP3 in causing immune escape from CD8+ T cell-mediated killing through a decrease in PD-L1 ubiquitination and subsequent proteasomal degradation by stabilizing OTUB1 mRNA in a PKP3-dependent manner. This work sheds light on a novel mechanism of PD-L1 regulation in NSCLC and provides a rationale to enhance the efficacy of anti-PD-1 treatment in NSCLC.

中文翻译：

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N6-甲基腺苷修饰的 circIGF2BP3 通过促进非小细胞肺癌中 PD-L1 的去泛素化抑制 CD8+ T 细胞反应以促进肿瘤免疫逃避

深入了解肿瘤中的免疫逃避机制对于克服耐药性和实现免疫治疗的创新进展至关重要。环状 RNA (circRNA) 与癌症进展有关。然而，关于 circRNA 是否会影响非小细胞肺癌 (NSCLC) 的免疫逃逸，仍有许多未知之处。我们进行了生物信息学分析，以分析和鉴定介导 NSCLC 中免疫逃避的 circRNA。进行荧光素酶报告基因分析、RNA 免疫沉淀 (RIP)、RNA pulldown 分析和荧光原位杂交，以鉴定 circIGF2BP3、miR-328-3p、miR-3173-5p 和 plakophilin 3 (PKP3) 之间的相互作用。体外 T 细胞介导的杀伤试验和体内同系小鼠模型用于研究 circIGF2BP3 及其下游靶点 PKP3 在 NSCLC 抗肿瘤免疫中的功能作用。通过免疫沉淀、RIP 和泛素化测定探索 PKP3 诱导的 PD-L1 上调的分子机制。我们证明了 circIGF2BP3 (hsa_circ_0079587) 在 NSCLC 中的表达增加，并且与 CD8+ T 细胞浸润呈负相关。在功能上，升高的 circIGF2BP3 在体外使共培养的 T 细胞失活，并在免疫活性小鼠模型中损害抗肿瘤免疫，这种作用依赖于 CD8+ T 细胞。从机制上讲，METTL3 介导 circIGF2BP3 的 N6-甲基腺苷 (m6A) 修饰，并以依赖于 m6A 读取蛋白 YTHDC1 的方式促进其环化。circIGF2BP3 通过海绵化 miR-328-3p 和 miR-3173-5p 竞争性上调 PKP3 表达，从而损害癌症免疫反应。此外，PKP3 与 RNA 结合蛋白 FXR1 结合以稳定 OTUB1 mRNA，而 OTUB1 通过促进其去泛素化来提高 PD-L1 的丰度。肿瘤 PD-L1 缺失完全阻断了 circIGF2BP3/PKP3 轴对 CD8+ T 细胞反应的影响。circIGF2BP3/PKP3 的抑制增强了抗 PD-1 疗法在 Lewis 肺癌小鼠模型中的治疗效果。总的来说，PKP3/PD-L1 特征和浸润性 CD8+ T 细胞状态将 NSCLC 患者分层为不同的风险组。我们的研究结果揭示了 circIGF2BP3 通过以 PKP3 依赖性方式稳定 OTUB1 mRNA 来减少 PD-L1 泛素化和随后的蛋白酶体降解，从而导致免疫逃避 CD8+ T 细胞介导的杀伤。这项工作揭示了 NSCLC 中 PD-L1 调控的新机制，并为提高抗 PD-1 治疗在 NSCLC 中的疗效提供了依据。