N(6)-methyladenosine-modified circIGF2BP3 inhibits CD8(+) T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer.
N(6)-methyladenosine-modified circIGF2BP3 inhibits CD8(+) T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer.
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N（6）-甲基腺苷修饰的circIGF 2BP 3通过促进非小细胞肺癌中PD-L1的去泛素化来抑制CD 8（+）T细胞应答以促进肿瘤免疫逃避
DOI：
10.1186/s12943-021-01398-4
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发表时间：
2021-08-20
期刊：
影响因子：
37.3
通讯作者：
Zhou Y
中科院分区：
文献类型：
--
作者：
Liu Z;Wang T;She Y;Wu K;Gu S;Li L;Dong C;Chen C;Zhou Y
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是否影响非小细胞肺癌（NSCLC）的免疫逃逸仍有很多未知之处。我们进行了生物信息学分析，以分析和鉴定NSCLC中介导免疫逃避的circRNA。采用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等方法，对circIGF 2BP 3、miR-328- 3 p、miR-3173- 5 p和斑嗜蛋白3（PKP 3）之间的相互作用进行了研究。体外T细胞介导的杀伤试验和体内同基因小鼠模型用于研究circIGF 2BP 3及其下游靶点PKP 3在NSCLC抗肿瘤免疫中的功能作用。PKP 3诱导的PD-L1上调的分子机制通过免疫沉淀、RIP和泛素化分析进行了探索。我们证明circIGF 2BP 3（hsa_circ_0079587）在NSCLC中表达增加，并且与CD 8 + T细胞浸润呈负相关。在功能上，升高的circIGF 2BP 3使体外共培养的T细胞失活，并在免疫活性小鼠模型中损害抗肿瘤免疫力，这种作用依赖于CD 8 + T细胞。在机制上，胃L3介导circIGF 2BP 3的N6-甲基腺苷（m6 A）修饰，并以依赖于m6 A阅读蛋白YTHDC 1的方式促进其环化。circIGF 2BP 3通过吸收miR-328- 3 p和miR-3173- 5 p竞争性上调PKP 3表达，以损害癌症免疫应答。此外，PKP 3与RNA结合蛋白FXR 1结合以稳定OTUB 1 mRNA，OTUB 1通过促进其去泛素化来提高PD-L1丰度。肿瘤PD-L1缺失完全阻断了circIGF 2BP 3/PKP 3轴对CD 8 + T细胞应答的影响。在刘易斯肺癌小鼠模型中，circIGF 2BP 3/PKP 3的抑制增强了抗PD-1疗法的治疗功效。总体而言，PKP 3/PD-L1特征和浸润性CD 8 + T细胞状态将NSCLC患者分为不同的风险组。我们的研究结果揭示了circIGF 2BP 3通过以PKP 3依赖性方式稳定OTUB 1 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CD 8 + T细胞介导的杀伤的功能。这项工作揭示了NSCLC中PD-L1调节的新机制，并为增强抗PD-1治疗NSCLC的疗效提供了理论依据。在线版本包含补充材料，可通过10.1186/s12943-021-01398-4获得。
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. The online version contains supplementary material available at 10.1186/s12943-021-01398-4.
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