抑癌基因LKB1通过调控Exo70 N-连接糖基化抑制肺癌转移的机制研究

Tumor suppressor LKB1 can inhibit the metastasis of lung cancer. However, the exact mechanisms remain elusive. Our preliminary results showed that in those cancer cell lines, in which LKB1 was absent, exo70, an important member of the exocyst, is N-linked glycosylated. Further studies indicated that the glycosylation leads to the migration of lung cancer cells, and the glycosylation could be inhibited by LKB1,which indicated that LKB1 might inhibit the lung cancer cell migration via regulation of Exo70 glycosylation. In this project, firstly, in cell and animal models, by using point mutations, glycosylation modifications, RNA interferences and 3D cell invasion assay, we will study: 1) the roles of Exo70 glycosylation in exocyst assembly and cell movement and migration; 2) the molecular mechanisms involved in LKB1 regulated exo70 glycosylation, including phosphorylation and glycosylation enzymes regulation; 3) mechanisms involved in LKB1 regulated exo70 glycosylation and lung cancer cell migration. Then, we will study the relationship between LKB1 and exo70 glycosylation as well as exo70 glycosylation and patient pathological staging and prognosis in patient tissue samples. Based on the above study, we will screen the lead compounds targeting the exo70 glycosylation to inhibit lung cancer cell migration. This study will provide theoretical basis for the diagnosis, target therapy and related drug discovery of lung cancer.

研究表明抑癌基因LKB1可以抑制肺癌转移，具体机制有待深入探索。我们发现在LKB1缺失的癌细胞株中，胞外分泌复合体重要成员Exo70发生N-连接糖基化，此糖基化促进肺癌细胞转移，回补LKB1能够抑制Exo70糖基化，提示LKB1可能通过调控Exo70糖基化抑制肺癌细胞转移。本项目将首先在细胞和动物水平，运用点突变、糖基化修饰、RNA干扰、3D球体细胞侵袭分析等方法研究：1. Exo70 糖基化对胞外分泌复合体装配及细胞运动和迁移的影响；2. LKB1调控Exo70糖基化的分子机制，包括磷酸化以及糖基转移酶调控；3. LKB1调控Exo70糖基化如何影响肺癌细胞转移。进而在临床样本中分析LKB1缺失与Exo70 糖基化的相关性以及Exo70糖基化与肺癌病理分期以及生存预后的相关性。最后以Exo70糖基化为靶点筛选抗肺癌先导化合物。项目将为肺癌的诊断、靶向治疗以及相关靶点药物开发提供理论依据。

肺癌是全球发病率最高的癌症，给人民健康带来巨大的威胁。早期原位肺癌治愈率和预后均较为理想，但是一旦发生转移则预后差，五年生存率低于20%。因此明确肺癌转移机制，找到关键分子对肺癌的治疗具有重要意义。本项目旨在研究重要的蛋白激酶LKB1与胞外分泌复合体关键亚基Exo70在调控肿瘤转移中的作用机制以及LKB1对Exo70的调控机制。首先，我们发现 LKB1通过调节Exo70蛋白第五位氨基酸（Gln5）的一种新蛋白修饰（前期初步认为是N-连接糖基化，后续研究通过多种手段证实为转谷氨酰胺酶介导的酰胺化修饰）来抑制肺癌转移。在进一步的机制研究中，我们发现Exo70 Gln5位点的酰胺化修饰可以促进胞外分泌复合体的聚合，进而促进基质金属蛋白酶的分泌，从而增强肺癌细胞的侵袭能力。LKB1能结合并磷酸化Exo70，进而抑制转谷氨酰胺酶催化Exo70发生酰胺化修饰。我们还进一步从天然产物库中筛选到能抑制Exo70酰胺化修饰且具有抑制肺癌细胞转移能力的化合物斑蝥素，可能成为潜在的抗肺癌侵袭转移靶向小分子药物。项目明确了酰胺化修饰对Exo70功能的重要影响，为肺癌的诊断、靶向治疗以及相关靶点药物开发提供提供新策略。