CRL4-DCAF13泛素连接酶调节非小细胞肺癌转移及其分子机制

DCAF13 is one of conserved substrate receptors of the Cullin4-RING E3 ubiquitin ligase, and its expression and function in human carcinomas is largely unknown. By analyzing the TCGA data, we previously found that DCAF13 is highly overexpressed and associated with poor prognosis in lung cancer including adenocarcinoma (AC) and squamous cell carcinoma (SCC). We further showed that Dcaf13 promotes cell migration using the disseminated mouse lung cancer cells generated from the KrasLSL-G12D/+;Trp53flox/flox mice and demonstrated that the oncogenic effect is dependent on its target substrates. Thus, we hypothesize that Dcaf13 plays an important role in lung cancer metastasis. To test our hypothesis, we will first determine if the differential expression of Dcaf13 affects lung cancer cell migration and invasion. To further elucidate the mechanism by which Dcaf13 regulates cell invasion and metastasis in lung cancer , we will combine diverse molecular biology techniques to identify its potential key substrates and characterize their roles in regulating metastasis ; We will further characterize the role and mechanism of Dcaf13 in lung cancer metastasis using xenograft and spontaneous lung cancer model and predict its correlation with patient prognosis by determining DCAF13 expression in clinical specimens. This study will identify a novel mechanism of regulation of metastasis by CRL4-DCAF13 in non-small cell lung cancer, providing a novel therapeutic target for inhibiting lung cancer metastasis.

DCAF13蛋白是cullin 4-RING泛素连接酶（CRL4）的底物识别受体之一，其在肿瘤中的作用迄今未明。我们前期通过TCGA数据库分析发现DCAF13不但在肺腺癌和肺鳞癌中高表达，而且与患者预后密切相关；后续实验以小鼠播散性肺腺癌细胞为模型，证实Dcaf13具有促进或维持肺癌细胞体外迁移的潜能，这种促癌效应与其下游特定底物密切相关。我们据此推测DCAF13具有促进肺癌远端转移的作用。为了证实这一假说，本项目拟从细胞水平鉴定DCAF13差异表达对肺癌细胞侵袭与迁移的影响；综合多重实验技术探索DCAF13通过调节其关键底物促进转移的分子机制；联合移植瘤和原发性肿瘤模型验证Dcaf13在肿瘤转移中的作用及机制；结合临床标本评价DCAF13表达与疾病预后相关性。本项目完成预期能够鉴定一种以CRL4-DCAF13为基础的非小细胞肺癌转移调节新机制，为临床抗肿瘤转移治疗及疾病预后提供科学依据。

肺癌是世界性高发、高度致死性恶性肿瘤。大部分患者最终死于肿瘤转移。因此，控制肿瘤转移是阻止患者病情恶化、提高疗效的关键措施。但迄今为止，临床上仍然缺乏特异、有效的抗肿瘤转移治疗方法。为了发现一种以cullin 4-RING泛素连接酶(CRL4)为基础的非小细胞肺癌转移调节新机制及潜在干预策略，本研究聚焦CRL4的底物识别受体家族蛋白，通过RNA干扰筛选策略及细胞生物学实验发现DCAF13具有促进肺癌细胞迁移及肿瘤细胞干性的生物学效应；我们通过小鼠肿瘤转移模型证实DCAF13表达下调能够有效抑制肿瘤细胞远处转移。与临床相关的是，我们联合TCGA数据库、cDNA芯片及肺癌组织芯片分析发现DCAF13在肺癌组织内异常高表达，并与患者生存期呈显著负相关，揭示了DCAF13表达异常与肿瘤转移的密切关联。在机制上，我们证实DCAF13能够通过非降解功能促进核糖体生物合成相关蛋白DDX47（一种DNA解旋酶）的泛素化修饰，并成功鉴定了泛素化修饰的赖氨基酸位点。DDX47泛素化位点突变后被发现能够有效抑制肿瘤细胞远处转移。在此基础上，我们进一步通过翻译组测序技术证实DCAF13能够通过介导DDX47解旋酶泛素化促进下游一系列促肿瘤转移分子的翻译。上述研究结果揭示了CRL4-DCAF13泛素连接酶通过调节肿瘤转移相关分子翻译促进肺癌转移的分子机制及潜在干预策略，达到预期研究目标。