Chen Zhi-Qiang, Zuo Xue-Liang, Cai Juan, Zhang Yao, Han Guo-Yong, Zhang Long, Ding Wen-Zhou, Wu Jin-Dao, Wang Xue-Hao
Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, NHC Key Laboratory of Liver Transplantation, Nanjing, 210029, China.
Department of Gastrointestinal Surgery, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu, 241001, China.
Exp Hematol Oncol. 2023 Feb 6;12(1):17. doi: 10.1186/s40164-023-00378-2.
Hypoxia is a hallmark of cancer, and is closely intertwined with tumor immune evasion. Circular RNAs (circRNAs) have been implicated in tumor response to immune checkpoint blockades. However, hypoxia-associated circRNAs that orchestrate the association between hypoxia and response to immunotherapy remain poorly understood. Here, we aimed to determine the roles of hypoxia-associated circRNAs in immune escape of hepatocellular carcinoma (HCC) cells.
Differentially expressed hypoxia-associated circRNAs were determined using high-throughput sequencing technology. HCC patients treated with PD-1 blockade were enrolled to assess the clinical significance of circPRDM4. RT-qPCR, western blotting, flow cytometry, T cell-mediated tumor cell killing assay, and enzyme linked immunosorbent assay were used to investigate the roles of circPRDM4 in immune escape of HCC cells in vitro. Patient-derived xenograft mouse models and adoptive human tumor infiltrating lymphocyte-CD8 T cell transfer were adopted to evaluate the effects of circPRDM4 in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification, dual-luciferase reporter assays, dot blotting, DNA in situ hybridization, and immunoprecipitation were utilized to examine the interaction between circPRDM4, HIF-1α, and CD274 promoter.
We identified circPRDM4 as a hypoxia-associated circRNA in HCC. circPRDM4 was upregulated in responders to PD-1 blockade and associated with therapeutic efficacy. In vitro and in vivo experiments showed that circPRDM4 induced PD-L1 expression and promoted CD8 T cell-mediated immune escape under hypoxic conditions. Mechanistically, circPRDM4 acted as a scaffold to recruit HIF-1α onto CD274 promoter, and cemented their interaction, ultimately promoting the HIF-1α-mediated transactivation of PD-L1.
These findings illustrated that circPRDM4 promoted immune escape of HCC cells by facilitating the recruitment of HIF-1α onto the promoter of CD274 under hypoxia, thereby inhibiting CD8 T cell infiltration in the tumor microenvironment. This work may provide a novel prognostic biomarker and therapeutic candidate for HCC immunotherapy.
缺氧是癌症的一个标志，并且与肿瘤免疫逃逸密切相关。环状RNA（circRNA）已被证明与肿瘤对免疫检查点阻断的反应有关。然而，协调缺氧与免疫治疗反应之间关系的缺氧相关circRNA仍知之甚少。在此，我们旨在确定缺氧相关circRNA在肝细胞癌（HCC）细胞免疫逃逸中的作用。
使用高通量测序技术确定差异表达的缺氧相关circRNA。纳入接受PD-1阻断治疗的HCC患者以评估circPRDM4的临床意义。采用RT-qPCR、蛋白质印迹、流式细胞术、T细胞介导的肿瘤细胞杀伤试验和酶联免疫吸附试验来研究circPRDM4在体外HCC细胞免疫逃逸中的作用。采用患者来源的异种移植小鼠模型和过继性人类肿瘤浸润淋巴细胞-CD8 T细胞转移来评估circPRDM4在体内的作用。利用RNA下拉、质谱分析、RNA免疫沉淀、染色质免疫沉淀、RNA纯化染色质分离、双荧光素酶报告基因检测、斑点印迹、DNA原位杂交和免疫沉淀来检测circPRDM4、HIF-1α和CD274启动子之间的相互作用。
我们确定circPRDM4是HCC中一种缺氧相关的circRNA。circPRDM4在PD-1阻断治疗的反应者中上调，并与治疗效果相关。体外和体内实验表明，circPRDM4在缺氧条件下诱导PD-L1表达并促进CD8 T细胞介导的免疫逃逸。机制上，circPRDM4作为支架将HIF-1α募集到CD274启动子上，并巩固它们之间的相互作用，最终促进HIF-1α介导的PD-L1反式激活。
这些发现表明，circPRDM4通过促进缺氧条件下HIF-1α募集到CD274启动子上，从而抑制肿瘤微环境中CD8 T细胞浸润，促进HCC细胞的免疫逃逸。这项工作可能为HCC免疫治疗提供一种新的预后生物标志物和治疗候选物。
