ANKMY2通过促进AC3降解调节肺癌发生的研究

Lung cancer isthe most common cancer both in China and in the world. Previous studies found that AC3 and cAMP signaling plays critical role in tumorigenesis. However, how AC3 is degraded and the role of AC3 degradation may play in lung caner development are still illusive. The function of ANKMY2 is still mysterious. Kaplan-Meier survival analysis found that the higher expression of ANKMY2 and the better overall survival in 2437 human lung cancer cases suggested that ANKMY2 might function as a tumor-suppressor; conversely, the higher expression of AC3 and the worse overall survival of lung cancer patients. ANKMY2 and AC3 may play critical role in lung cancer tumorigenesis. Our preliminary data suggested that ANKMY2 binds to AC3 and promotes AC3 degradation. Thus ANKMY2 may be important for lung caner development. We are planning to investigate the underline mechanism through the following assays: 1) test whether ANKMY2 and AC3 interact physically using co-IP, GST pull-down and immunofluoresence assays, and further mapping the interacting domain of ANKMY2 and AC3; 2)next, ANKMY2 would be over-expressed or knocked down in lung cancer cell lines, analyzing the underlining mechanism of AC3 degradation and its impact on lung cancer development; 3)AC3 degradation would also be examined in ANKMY2 knockout mice; 4) furthermore, a stable cell line in which ANKMY2 was knocked down by shRNA would established; the cells were then used in colony formation in soft agar assays and tumor formation experiments in nude mice to investigate the role ANKMY2 in lung cancer development. This project would reveal new function of ANKMY2 and how the function is carried out. This project would also provide a new target for cancer therapy against lung cancer.

肺癌是我国发病率和死亡率均居首位的癌症。研究表明AC3及cAMP信号在肿瘤的发生和发展中起重要作用。但AC3的降解的调节机制尤其是它在肺癌发生中的作用尚未见研究。ANKMY2的功能尚不明确。我们发现，ANKMY2高的肺癌病人的生存时间长，而AC3高的病人的生存时间短，ANKMY2和AC3可能调节肺癌的发生；ANKMY2与AC3相互作用并促进AC3的降解，可能调节AC3的降解。项目拟： 1）通过co-IP等实验验证二者的相互作用，并确定相互作用的结构域；2）在肺癌细胞系中过表达ANKMY2或敲减ANKMY2，分析其对AC3的降解的调节作用及可能的机制，并探讨其对肿瘤形成的影响；3）分析ANKMY2敲除小鼠中AC3的降解；4）分析ANKMY2敲减细胞在裸鼠皮下成瘤情况，在体内研究其在肿瘤发生中的作用。本项目可能揭示ANKMY2蛋白的功能并阐明该功能发挥作用的机制，为肺癌治疗提供新的靶点。

cAMP 是第一个被发现的第二信使。它由腺苷酸环酶(adenylyl cylcase, ADCY, AC)催化 ATP 分解得到。细胞外的信号通过受体-GPCR-AC-cAMP 途径在动物的整个生命过程中从胚胎发育到细胞凋亡，方方面面发挥重要作用。我们之前的研究发现 TRIP-Br1 与 XIAP E3 泛素连接酶形成复合体，调节多种腺苷酸环化酶的降解。但是 AC3 的降解并不受这一复合体的调节，其降解机制尚不清楚。在本研究中，我们发现 ANKMY2 的氮端能够与 AC3 结合，并调节AC3 的总体表达水平以及细胞膜上的表达水平。我们通过 CHX chase 实验发现，ANKMY2 与 AC3 结合后可以加快该蛋白的降解速率。而进一步对 AC3 的 泛素化进行分析发现，ANKMY2 能够促进 AC3 的泛素化。我们对病人的生存时间和 ANKMY2,AC3 的表达水平做了 Kaplan-Meier survival plot。跟我们蛋白方面的研究结果一致的是，我们发现 AC3 表达水平高的病人生存时间较短，而ANKMY2 表达水平高的病人生存时间较长。人的小细胞肺癌细胞系H1299中敲减ANKMY2会抑制细胞生长。综上，我们发现 ANKMY2 结合到AC3 上，并通过促进 AC3 的泛素化来调节其表达水平，以及调节小细胞肺癌细胞的生长。