NEDD4介导PTEN泛素化降解在NSCLC EGFR-TKI耐药中的作用及调控机制研究

PTEN，a tumor suppressor gene with phosphatase activity, has been found to play an important role in cell proliferation, apoptosis and chemoresistance. Our previous study found that PTEN protein levels were significantly reduced in erlotinib-induced resistant HCC827/ER cells. The ubiquitination-mediated degradation was the main reason, but the exact mechanism is unknown. We use H1975, HCC827 and erlotinib-resistant cells H1975/ER and HCC827/ER in this study to address that the decreased PTEN results in relative activation of PI3K /AKT/mTOR pathway, which in turn plays an important role in the induction of resistance; to demonstrate NEDD4-mediated PTEN ubiquitination and degradation using MG132 inhibit ubiquitination, immunoprecipitation, immunoblotting, NEDD4 knockdown or overexpression and xenograft experiments; to indicate the role of miR-30b/c in regulating NEDD4-mediated PTEN ubiquitination and degradation in the EGFR-TKIs induced drug resistance with bioinformatic prediction, dual luciferase assay and inhibitors or overexpression experiments, which will be verify with clinical specimens. This project will be helpful for further understanding of the.mechanisms underlying NSCLC EGFR-TKI resistance, and provide new ideas and targets for the treatment of NSCLCs.

PTEN作为抑癌基因，在细胞增殖、凋亡和化疗耐药等过程中都有重要作用。我们前期研究发现厄洛替尼诱导的耐药细胞HCC827/ER PTEN蛋白水平显著降低；且泛素化降解是其主要原因，但具体机制不明。本项目以H1975和HCC827及其耐厄洛替尼细胞H1975/ER和HCC827/ER为研究对象，证实PTEN下降，减少对PI3K/AKT/mTOR通路的抑制，在耐药性诱导中起重要作用；通过MG132泛素化抑制、免疫共沉淀结合免疫印迹、NEDD4敲低或过表达及其相应移植瘤实验，证实NEDD4泛素化降解PTEN的作用；通过生物信息学预测、双荧光素酶实验及抑制剂或过表达等，证明miR-30b/c调控NEDD4降解PTEN在NSCLC EGFR-TKI继发耐药中的作用，并经临床标本检测、验证。本项目有助于深入认识NSCLC EGFR-TKI继发耐药机制，并提供新的干预靶点和思路。

作为抑癌基因，PTEN在细胞增殖、凋亡和化疗耐药中有重要作用。研究表明，E3泛素连接酶NEDD4（Neural precursor cell expressed developmenttally down-regulated 4，神经前体细胞表达下调因子4，也叫NEDD4-1）能通过泛素化和蛋白水解作用负调控PTEN蛋白表达。我们前期研究发现厄洛替尼诱导的耐药细胞HCC827/ER PTEN蛋白水平显著降低；且泛素化降解是其主要原因，但具体机制不明。本项目以含EGFR19del突变的HCC827及其厄洛替尼诱导的耐药细胞HCC827/ER为研究对象，探讨NEDD4和PTEN在厄洛替尼继发耐药中的作用。研究发现：HCC827/ER PTEN下调表达，NEDD4上调表达，呈负相关；敲低HCC827/ER中NEDD4表达后，PTEN和p-AKT的表达上升，HCC827/ER对厄洛替尼的敏感性部分恢复，且能够改善NSCLC荷瘤小鼠对厄洛替尼继发耐药。在H1650/ER细胞（含有EGFR19del突变和PTEN缺失）中，敲低NEDD4，p-AKT的表达和细胞对厄洛替尼的敏感性无明显变化；而在敲低NEDD4的细胞中转入PTEN后，则p-AKT表达升高，细胞对厄洛替尼的敏感性增加。目前正通过生物信息学预测和small RNA测序等，筛选调控NEDD4的miRNA（s）。综上所述，NEDD4通过促进PTEN泛素化降解，减少对PI3K/AKT/mTOR通路的抑制，诱导NSCLC厄洛替尼继发耐药。本项目有助于深入认识NSCLC EGFR-TKI继发耐药机制，并提供新的干预靶点和思路。