ING5抑制Ku70 T455磷酸化在其抗肺癌侵袭转移中的作用和机制研究

Lung cancer is the leading cause of malignant cancer mortality. Inhibiting cancer invasion and metastasis is crucial to improve survival of lung cancer patients. Ku70 is a typical damage repair protein and regulator of apoptosis. Recenty, it has been found to be a promising target for treatment of cancer invasion, suggesting that it also play an important role in cancer invasion and metastasis. Previously, we have found that the Inhibitor of Growth (ING) family member ING5 could significantly inhibit Epithelial-Mesenchymal Transition (EMT) and invasion and metastasis of lung cancer cells. ING5 also participates in regulation of protein acetylation and phosphorylation. ING5 overexpression could significantly downregulate phosphorylation of Ku70 at Threonine (T) 455 accompanied with decreased Ku70 protein level. Furthermore, Ku70 siRNA reversed ING5 knockdown-promoted invasion of lug cancer cells. These results suggest that ING5 may regulate Ku70 function at post-translational modification level to inhibit lung cancer progression. What are the significance and mechanisms of downregulated phosphorylation of Ku70T455 by ING5? In the current project, we are going to perform experiments including gene knockdown and rescue, protein kinase siRNA library and et al. to investigate decreased phosphorylation of Ku70 at T455 by ING5 and its regulation of ubiquitin-dependent degradation of Ku70. The aims of the study are to define the effects of Ku70T455 phosphorylation on ING5 function and explore the mechanisms by which ING5 downregulates Ku70T455 phosphorylation. Our study would reveal new mechanisms of how ING5 inhibits lung cancer invasion and provide treatment targets for lung cancer metastasis.

肺癌是恶性肿瘤导致死亡的首要原因，提高患者生存率的重要措施是抑制肺癌侵袭转移。Ku70是经典的损伤修复和凋亡调控分子，最近发现其可作为抗肿瘤侵袭治疗靶点，提示Ku70在肿瘤的侵袭转移过程中亦发挥重要作用。我们前期研究发现生长抑制因子ING5可显著抑制肺癌EMT和侵袭转移，并参与调控蛋白的乙酰化和磷酸化修饰，ING5过表达可显著抑制Ku70T455磷酸化并伴有Ku70蛋白水平下调，Ku70基因敲减可抑制ING5下调引起的肺癌细胞高侵袭性。提示ING5可能在翻译后修饰水平调控Ku70而发挥抑癌作用。ING5抑制Ku70T455磷酸化的意义和机制如何？本课题拟采用基因敲减回补和siRNA文库等研究ING5抑制Ku70T455磷酸化及其对Ku70泛素化降解的调控，探讨该位点磷酸化对ING5抑癌作用的影响和ING5下调该位点磷酸化机制，进而揭示ING5抑制肺癌侵袭转移新机制，为肺癌转移治疗提供靶点。

肺癌是恶性肿瘤导致死亡的首要原因，提高患者生存率的重要措施是抑制肺癌侵袭转移。Ku70是经典的损伤修复和凋亡调控分子，最近研究发现其可作为抗肿瘤侵袭治疗靶点，提示Ku70在肿瘤的侵袭转移过程中亦发挥重要作用。我们前期研究发现生长抑制因子ING5可显著抑制肺癌细胞的EMT和侵袭转移，并参与调控靶蛋白的乙酰化和磷酸化修饰，ING5过表达可显著抑制Ku70 T455位点磷酸化并伴有Ku70蛋白水平下调，Ku70基因敲减可抑制ING5下调引起的肺癌细胞高侵袭性，提示ING5可能在翻译后修饰水平调控Ku70而发挥抑癌作用。基于此，为深入研究ING5抑制肺癌侵袭转移的分子机制，本研究以肺癌A549细胞和H1299细胞ING5过表达细胞为研究对象，综合采用基因转染、免疫共沉淀、点突变和基因敲减回补技术构建了Ku70稳定敲减（KD）以及敲减回补T455D野生型（WT）和T455A磷酸化缺陷（Mut）细胞株，细胞水平通过采用CCK8细胞增殖和克隆形成实验、Wound-healing实验、迁移和侵袭实验、qRT-PCR和western blotting方法证实，ING5可显著抑制Ku70 T455位点磷酸化，并降低Ku70的稳定性和介导其泛素降解，而T455磷酸化位点突变能够逆转该作用。此外，动物水平通过构建裸鼠皮下种植瘤模型和尾静脉注射肿瘤细胞转移瘤模型，证实ING5过表达通过抑制Ku70 T455位点磷酸化进而抑制肺癌细胞的体内成瘤和转移，并大大延长裸鼠生存时间，而T455磷酸化位点突变能够逆转ING5过表达介导的体内抑制肺癌细胞的生长和转移作用。综上所述，本研究初步阐明了抑癌基因ING5通过抑制Ku70蛋白T455位点磷酸化降低Ku70蛋白稳定性，进而下调Ku70蛋白水平，发挥显著的抑制肺癌侵袭和转移作用新机制，并揭示了T455位点磷酸化对原癌基因Ku70促癌功能的影响和意义，为ING5抑癌作用机制作新的补充，同时为临床靶向Ku70的抗肺癌侵袭转移治疗提供依据理论依据和作用靶点。

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