Xu Hongbo, Cai Feng, Xu Lu, Jiang Dengsheng, Wang Gengming, Zhang Xianwen, Zhang Yajun
Department of Radiation Oncology, The First Affiliated Hospital of Bengbu Medical University Zhihuai Road, Bengbu 233000, Anhui, China.
Am J Cancer Res. 2025 Aug 15;15(8):3449-3459. doi: 10.62347/BIQP4822. eCollection 2025.
Radiotherapy is a cornerstone treatment for lung cancer; however, enhancing its efficacy and overcoming immune escape mechanisms - particularly those mediated by tumor-associated macrophages (TAMs) expressing programmed death-ligand 1 (PD-L1) - remain significant challenges. The E3 ubiquitin ligase RNF200 has been implicated in the regulation of PD-L1 expression, yet its role in the context of radiotherapy is not well understood. To address this, non-small cell lung cancer (NSCLC) tissue samples from patients with and without prior radiotherapy were analyzed for RNF200 and PD-L1 expression using quantitative RT-PCR and Western blotting. Additionally, RAW264.7 macrophages were subjected to ionizing radiation and genetically manipulated to assess the impact of RNF200 on PD-L1 expression and stability through co-immunoprecipitation and ubiquitination assays. Co-culture experiments with macrophages and lung cancer cells were performed to evaluate the influence of RNF200 on radiotherapy sensitivity. In NSCLC tissues and macrophages, radiotherapy was found to downregulate RNF200 expression while upregulating PD-L1 expression. Overexpression of RNF200 led to marked suppression of PD-L1 expression, whereas RNF200 knockdown produced the opposite effect. Co-immunoprecipitation and ubiquitination assays revealed that RNF200 physically interacted with PD-L1 and promoted its polyubiquitination and proteasomal degradation. Furthermore, co-culture studies demonstrated that macrophages overexpressing RNF200 enhanced the sensitivity of lung cancer cells to radiotherapy, as evidenced by reduced proliferation, increased necrosis, and decreased secretion of transforming growth factor beta TGF-β. Collectively, these findings indicate that RNF200 enhances radiotherapy sensitivity in lung cancer by regulating PD-L1 expression through ubiquitination. Targeting RNF200 may represent a promising strategy to improve the efficacy of radiotherapy in lung cancer treatment.
放射治疗是肺癌的基石性治疗方法；然而，提高其疗效并克服免疫逃逸机制——尤其是由表达程序性死亡配体1（PD-L1）的肿瘤相关巨噬细胞（TAM）介导的免疫逃逸机制——仍然是重大挑战。E3泛素连接酶RNF200与PD-L1表达的调控有关，但其在放射治疗背景下的作用尚不清楚。为了解决这个问题，使用定量RT-PCR和蛋白质印迹法分析了有或无放疗史患者的非小细胞肺癌（NSCLC）组织样本中的RNF200和PD-L1表达。此外，对RAW264.7巨噬细胞进行电离辐射并进行基因操作，通过免疫共沉淀和泛素化测定评估RNF200对PD-L1表达和稳定性的影响。进行了巨噬细胞与肺癌细胞的共培养实验，以评估RNF200对放射治疗敏感性的影响。在NSCLC组织和巨噬细胞中，发现放射治疗下调RNF200表达，同时上调PD-L1表达。RNF200的过表达导致PD-L1表达明显受到抑制，而RNF200基因敲低则产生相反的效果。免疫共沉淀和泛素化测定表明，RNF200与PD-L1发生物理相互作用，并促进其多聚泛素化和蛋白酶体降解。此外，共培养研究表明，过表达RNF200的巨噬细胞增强了肺癌细胞对放射治疗的敏感性，表现为增殖减少、坏死增加以及转化生长因子β（TGF-β）分泌减少。总体而言，这些发现表明RNF200通过泛素化调节PD-L1表达来增强肺癌的放射治疗敏感性。靶向RNF200可能是提高肺癌放射治疗疗效的一种有前景的策略。
