泛素连接酶TRIM25介导的PTEN非典型多聚泛素化对非小细胞肺癌化学耐药的机制研究

Chemoresistance is a key obstacle in the treatment of non-small-cell lung cancers, which determines the therapeutic efficacy and clinical outcomes, therefore, how to overcome chemoresistance is critical in the treatment of NSCLC patients. It is well established that inactivation of the tumor suppressor gene PTEN is closely associated with the chemoresistance of NSCLC. But, the mechanism is unclear. In our previous study, we found that the ubiquitin ligase TRIM25 mediated PTEN polyubiquitination but without degradation, moreover, AKT and mTOR were highly activated in these cells, which was associated with the resistance of NSCLC cell line to cisplatin. These results suggest that TRIM25 mediates atypical polyubiquitination of PTEN thus inhibiting its suppression on the PI3K/AKT/mTOR signaling pathway and chemoresistance. In the present proposal, we will dissect the detailed mechanisms by designing a serial of mutants of TRIM25, PTEN and ubiquitin based on their functional domain or key amino acid residuals to characterize the interaction of TRIM25 and PTEN, as well as the ubiquitination of PTEN. We will also measure PTEN activity after ubiquitination by TRIM25 in cells and cell-free ubiquitination system. The effects of TRIM25/PTEN ubiquitination on proliferation, migration, survival, and resistance of NSCLC cell lines will also be evaluated. Finally, we will determine the expression level of TRIM25 and PTEN ubiquitination in primary NSCLC patient samples in association to the malignancy classification and clinical outcomes. The study will provide a solid frame for novel mechanism in NSCLC chemoresistance and drug discovery to overcome NSCLC chemoresistance.

化学耐药性是非小细胞肺癌（NSCLC）治疗中的常见问题，决定着NSCLC的疗效和预后，克服耐药性因而成为NSCLC治疗的关键。抑癌蛋白PTEN的失活与NSCLC的耐药性密切相关，但机制不清楚。我们最近发现NSCLC细胞中泛素连接酶TRIM25能诱导PTEN的多聚泛素化，但PTEN并不发生降解，而使AKT和mTOR信号却被激活，提示TRIM25通过非典型的泛素化修饰抑制了PTEN的活性从而参与NSCLC的化学耐药。本项目拟在前期研究的基础上通过基因突变以及细胞内和无细胞泛素化系统分析TRIM25介导PTEN发生泛素化的具体特征和分子机制，然后在细胞、动物和NSCLC患者肿瘤样本中分析TRIM25介导PTEN泛素化对NSCLC化学耐药的影响和机制，为进一步阐述NSCLC 细胞的化学耐药机制和发现新的能克服NSCLC耐药性的化学药物奠定坚实的基础。

非小细胞肺癌（NSCLC）是一种快速进展的肺部恶性肿瘤，是目前国内外癌症相关死亡的主要原因。PI3K催化合成PI(4,5)P2中的PI(3,4,5)P3以及由此产生的PI(3,4,5)P3，然后作为第二信使导致AKT和mTOR激酶的激活，从而促进NSCLC细胞的增殖、侵袭和细胞内运输。PI(3,4,5)P3可以被PTEN 的特定磷酸酶水解为PI(4,5)P2。因此PTEN可以拮抗AKT的激活，抑制PI3K/AKT/mTOR信号通路。然而，PTEN/AKT/mTOR信号通路在NSCLC中经常发生失调，但其机制尚不清楚。.本研究主要探讨TRIM25介导PTEN的多聚泛素化及具体机制，TRIM25介导的PTEN泛素化对PI3K/AKT/mTOR信号通路的影响，TRIM25介导PTEN非典型性泛素化对NSCLC细胞的化学耐药的影响。.本研究发现泛素连接酶TRIM25在NSCLC组织中高表达，与NSCLC患者的不良预后呈负相关，TRIM25促进NSCLC细胞生存和肿瘤生长。机制研究表明，TRIM25通过B-box、CC和SPRY域与PTEN相互作用，并介导PTEN与K63连接的K266位点多泛素化。这种修饰可以防止PTEN的质膜移位，降低其磷酸酶活性，从而积累PI(3,4,5)P3，因此TRIM25激活了AKT/mTOR信号，抑制TRIM25可增强NSCLC的化疗敏感性。此外，我们发现抗菌药硝羟喹啉（Nitroxoline，NXQ）可以通过减少其K63-连接的多泛素化激活PTEN，并使NSCLC对CDDP诱导的凋亡敏感。本研究发现了TRIM25对PTEN信号通路的一种新调控，TRIM25/PTEN/AKT/mTOR可能是NSCLC治疗的一个有前途的靶点。