靶向蛋白Neddylation修饰通路诱导非小细胞肺癌放射增敏及其机制研究

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer fail treatment. Proteasome inhibition has demonstrated radiosensitizing properties in non-small cell lung cancer model and in association with accumulation of cullin-RING E3 ligases (CRLs) substrate (e.g. IκBα) involved in DNA damage and repair. These suggest that CRLs may serve as a direct radiosensitizing target in this human cancer. Given that CRLs activation is exclusively dependent on cullin neddylation, we reasoned that inhibition of the neddylation pathway may improve radiotherapy in non-small lung cancer. To test this hypothesis, we will first determine the effect of the neddylation pathway inhibition by genetic (RNAi) and pharmacological (MLN4924, an investigational small molecule inhibitor of neddylation entering Phase I/II clinical trials) approaches on radiosensitization of non-small cell lung cancer using cell culture model.For better understanding of the mechenisms for radiosensitization by targeting the neddyhlation pathway, we use RNAi library to identify CRLs substrates and investigate their roles in regulating this biological phenotype. To predict human clinical efficacy of targeting the neddylation pathway by MLN4924, we will further adapt primary mouse lung cancer triggered by mutation in K-ras (K-rasG12D ) in combination with p53 null deletion. Tumor response of mice to radiation therapy will be assessed with micro-CT and FLT-PET and the mechanistic study is to be carried out in-dept. Collectively，we proposed thatthe neddylayion pathway may be a promising target for the improvement of local therapy in non-small cell lung cancer.

泛素-蛋白酶体系统已被证实是非小细胞肺癌的放射增敏靶标，其增敏机制与Cullin-RING E3 泛素连接酶（CRLs）底物积聚密切相关。基于CRLs活化高度依赖Neddylation修饰这一生物学特征，我们设想Neddylation修饰通路可以作为非小细胞肺癌放射增敏的直接靶标。本研究首先利用RNA干扰技术和药理学干预方法，从体外细胞水平评价靶向Neddylation修饰通路诱导非小细胞肺癌放射增敏的效果；然后利用RNA干扰文库筛选肺癌放射增敏相关的CRLs底物并阐明其在肺癌放射增敏中的作用，以揭示放射增敏的分子机制。为了模拟肿瘤在正常肿瘤微环境下对放射治疗的反应，我们拟用小鼠原发性肺腺癌作为临床前治疗模型，深度研究靶向Neddylation修饰通路诱导肺癌放射增敏的效果及机制。本项目的完成将为今后临床通过靶向Neddylation修饰通路提高非小细胞肺癌的放射治疗效果提供科学依据。

肺癌是当今死亡率最高的恶性肿瘤，其中非小细胞肺癌病例占80%以上。因其具有高度异质性与侵袭性，患者往往预后差、肿瘤易复发并产生治疗抵抗。基于早期研究基础，我们推测蛋白Neddylation修饰通路与非小细胞肺癌放射敏感性密切相关，并申请立项，旨在鉴定蛋白Neddylation修饰通路是非小细胞肺癌的放射增敏靶标，并阐明其作用分子机制。我们联合运用过表达、RNA干扰、免疫沉淀、质谱分析、报告基因和基因编辑等多种技术手段，系统研究蛋白Neddylation修饰通路及其下游关键通路在调控非小细胞肺癌放化疗敏感性中的作用及分子机制。我们研究结果发现：蛋白Neddylation修饰通路的活性差异与非小细胞肺癌放射敏感性密切相关，如抑制Neddylation修饰通路活性能够提高肺癌细胞的放射敏感性，而提高Neddylation修饰通路活性则促进肺癌细胞放射抵抗。鉴于Neddylation修饰通路关键性底物CRL4泛素连接酶对DNA损伤修复及细胞周期进展具有重要调节作用，我们聚焦CRL4泛素连接酶家族并发现其中CRL4-DCAF12泛素连接酶能够通过非降解方式调节肿瘤细胞CCT蛋白折叠机器（一种分子伴侣素）和经典抗调亡蛋白XIAP的泛素化，从而影响肿瘤细胞存活及放化疗敏感性。此外，我们证实DCAF12泛素连接酶在肺腺癌组织内异常高表达，其表达水平与患者预后呈显著负相关。这些研究结果揭示了“Neddylation-CRL4-DCAF12”信号轴在肺癌组织中异常活化的病理学意义及该信号轴可作为非小细胞肺癌治疗的潜在干预靶点。本项目发表重要专著论文1篇，申请专利3项，培养研究生4名。