N4BP3抑制PTEN通路促进乳腺癌细胞血行转移的作用和分子机制研究

Metastasis is the main cause of death in breast cancer, but the molecular mechanism of metastasis remains largely unknown. Based on our established CTCs isolation technique, we found that N4BP3 was highly expressed in the CTCs of metastatic breast cancer. Our pilot experiment results showed that N4BP3 can target PTEN which is important in PI3K/AKT signaling pathway, and significantly regulate the ability of proliferation, invasion and metastasis in breast cancer cells. Analysis of TCGA public databases indicated that N4BP3 was inversely correlated to the distant metastasis free survival of breast cancer. N4BP3 was known as a potential adaptor protein of NEDD4, which is the key E3 ubiquitin protein ligase for PTEN. Until now, the function of N4BP3 on breast cancer metastasis has not been reported in the literature. Thus, in this project, we will investigate the role of N4BP3 in the hematogenous metastasis of breast cancer by in vitro and in vivo experiments, and explore the mechanism of N4BP3 inhibiting PTEN from two aspects: One is that N4BP3 bound to the NEDD4 and caused the PTEN ubiquitination degradation, the other is that N4BP3 functioned on the PTEN promoter and inhibited the PTEN transcription. Finally, validation will be performed in clinical breast cancer patients and mouse model. This work aims to explore the molecular mechanism of breast cancer hematogenous metastasis from aspect of CTCs and would contribute a solid foundation for the coming research into the target treatment for metastatic breast cancer.

远处转移是乳癌的主要死因，但转移的具体机制尚不清楚。我们利用前期建立的基于表面抗原的循环肿瘤细胞（CTCs）捕获技术发现了一个转移性乳癌CTCs中高表达的新分子N4BP3，前期实验发现N4BP3能促进乳癌细胞增殖和转移能力，且能抑制PTEN的mRNA转录水平和蛋白表达。TCGA数据库提示N4BP3与乳癌远处转移和生存密切相关。文献报道N4BP3是PTEN重要泛素化连接酶NEDD4的潜在结合蛋白之一，但目前N4BP3在乳癌转移中的作用机制尚不清楚。本项目拟在验证N4BP3抑制PTEN从而促进乳癌转移作用的基础上，从N4BP3结合NEDD4而催化PTEN的多泛素化降解，或抑制PTEN启动子转录而下调PTEN产生这两方面来研究N4BP3抑制PTEN的分子机制，进而通过临床大样本和小鼠移植瘤模型验证。项目从CTCs这个新视点探究乳癌转移的关键分子机制，为靶向治疗转移性乳癌奠定扎实的研究基础。

乳腺癌是全球女性最高发的癌症，通过血行继而远处转移是乳腺癌的主要死因，目前对乳腺癌血行转移的具体机制尚不清楚。乳腺癌细胞具有较强的上皮间质转化的特点，我们基于乳腺癌表面抗原，建立能够同时捕获上皮型和间质型乳腺癌CTCs的平台（negFACS-IF:E/M）。基于该平台发现乳腺癌患者外周血CTCs的上皮-间质特征与乳腺癌的分期、分子分型、复发转移密切相关。在此基础上发现了一个转移性乳癌CTCs中高表达的新分子N4BP3，体内外实验发现N4BP3能够促进乳腺癌细胞的迁移和侵袭能力。TCGA数据库分析后发现乳腺癌组织中的N4BP3表达明显高于癌旁组织，且有远处器官转移的IV期乳腺癌中N4BP3表达量显著增高，通过GOBO数据库分析, 我们发现N4BP3的高表达在ER+和ER-的乳腺癌患者中都提示更差的无远处转移生存（DMFS）。探索其机制，发现N4BP3会引起乳腺癌细胞EMT相关蛋白的改变，且N4BP3亦可通过泛素化降解等相关途径对其下游蛋白进行调控。本项目建立的negFACS-IF:E/M平台作为新方法可用于临床乳腺癌复发转移预测的补充手段，同时N4BP3这个分子可能成为乳腺癌复发转移的潜在分子标志物。本研究从乳腺癌CTCs这个新视点探究乳腺癌转移的关键分子机制，为靶向治疗转移性乳癌奠定扎实的研究基础。