铜绿假单胞菌分泌型Ndk促进宿主非小细胞肺癌转移的分子机制研究

Pseudomonas aureginosa (P. aureginosa) is one of the most prevalent pathogens in lung infection, especially in patients suffered from non-small cell lung cancer (NSCLC). Researches have shown that the inhbition of the phosphorylation of mammalian ste20-like protein kinase 3 (MST3) on tyrosine 190 (T190) promotes tumor metastasis. In our previous study, we have found that the nucleoside diphosphate kinases (Ndk) secreted from P. aureginosa can be translocated into the host cells upon infection, and it interacts with the MST3, which results in the inhibition of the phosphorylation of MST3 on T190. This result indicates that P. aureginosa might promote tumor metastasis of NSCLC patients through the Ndk-mediated regulation of MST3. To verify this hypothesis, we firstly propose to identify the critical binding domains and sites between Ndk and MST3 by constructing the truncated and site-directed mutants. Secondly, we aim to clarify the effects on auto phosphorylation, kinase activity, sub-cellular location and protein stability of MST3, and on the tumor invasion and metastasis upon the interaction between Ndk and MST3. Thirdly, we set out to investigate the effect on tumor growth and metastasis upon P. aureginosa infection or Ndk treatment in the subcutaneous xenograft models of lung carcinoma. Lastly, we are intended to investigate the interaction pattern among Ndk, MST3 and NM23-H1 or NM23-H2 (the homologous protein of P. aureginosa Ndk), and the biological effects upon their interaction. This investigation might shed new light on the pathogenesis of P. aureginosa, and might also provide a new guidance for the prevention of tumor metastasis of NSCLC patients in clinic.

铜绿假单胞菌（PA）是肺部感染最常见病原菌之一，非小细胞肺癌（NSCLC）患者尤易感染。研究表明抑制MST3-T190位点自身磷酸化可促进肿瘤迁移。课题组前期发现，PA感染时其分泌型Ndk可进入细胞并与MST3互作，且伴有MST3-T190位点磷酸化水平降低，提示PA感染可能通过Ndk介导的MST3途径促进NSCLC患者肿瘤转移。本项目拟采用截断及位点突变等技术，考察Ndk与MST3互作关键区域及位点；考察该互作对MST3自身磷酸化、激酶活性、胞内分布、稳定性及对癌细胞侵袭、迁移的影响。构建肺癌皮下移植瘤动物模型，考察PA感染及Ndk蛋白对小鼠肿瘤生长、转移的影响。此外，考察人Ndk同源蛋白NM23-H1/H2与MST3互作方式及生物学效应。结果将对PA致病性的认识提供全新资料，并为临床NSCLC患者尽量避免PA感染，控制肿瘤转移，提供指导作用。

非小细胞肺癌（Non-small cell lung cancer，NSCLC）是人类呼吸系统最常见的恶性肿瘤，其发病率及死亡率在我国乃至全球高居前列。NSCLC细胞具有的极高侵袭和迁移能力是其导致肺癌复发以及抗癌治疗失败的关键因素。MST3是哺乳动物不育系20样激酶家族成员，与肿瘤的转移密切相关，但相关研究主要集中在乳腺癌和肠道肿瘤领域。为考察MST3在NSCLC发生、发展中的作用，本课题组首先通过对肿瘤数据库信息挖掘发现，MST3高表达与NSCLC病人的不良预后呈正相关。通过免疫组化染色证实，NSCLC病人肿瘤组织MST3表达明显高于癌旁组织。将MST3稳定干扰或过表达的肺腺癌细胞于小鼠皮下荷瘤证实，MST3高表达可促进肿瘤的生长，而干扰则呈现相反效应。通过蛋白组学、GO功能和KEGG通路富集分析发现，MST3高表达时差异表达蛋白主要富集在与肿瘤转移密切相关通路，如细胞粘附、骨架形成等。结合蛋白组学分析结果发现，MST3过表达可促进与肿瘤细胞转移密切相关的波形蛋白Vimentin S39磷酸化。因此，我们考察了MST3-Vimentin间相互联系在NSCLC细胞转移中的作用。通过干扰/过表达、Western blot、Transwell细胞迁移和划痕等实验证实，MST3可通过磷酸化Vimentin S39促进肿瘤细胞转移。另外，MST3过表达可促进AKT1 S124位点磷酸化，而干扰AKT1明显减少了MST3介导的Vimentin S39磷酸化，提示MST3可通过磷酸化AKT1增加Vimentin磷酸化，促进 NSCLC细胞转移。为进一步探讨MST3介导Vimentin S39磷酸化的可能机制，我们通过质谱、免疫共沉淀和免疫荧光等方法检测发现，MST3与Vimentin或MST3与AKT1具有分子间直接相互结合，提示MST3可通过与Vimentin直接互作或通过与AKT1互作，进而增加AKT1介导的Vimentin磷酸化，促进NSCLC细胞转移。上述结果揭示了MST3在NSCLC发生、发展中的作用及机制，其相关研究可为临床上NSCLC的治疗提供新的思路和作用靶点。. 在本课题的资助下，已发表SCI论文4篇，中文核心期刊4篇；申请国家发明专利1项（处于实审阶段），培养硕士研究生2名，博士研究生1名。