PRAS40在尤文肉瘤发生发展中的作用和分子机制研究

Ewing's sarcoma is a highly aggressive and highly metastatic tumor predominantly afflicting adolescents and young adults. With conventional treatment, the 5-year disease-free survival rate for patients with localized Ewing's sarcoma is only 60-70% and that for individuals with metastases drops to less than 20%. Thus, novel treatments are needed urgently. .EWS encodes an RNA-binding protein whose function remains largely unknown, whereas the chromosomal translocations, which fuse the N-terminal domain of EWS to the DNA-binding domain of the ETS family transcription factors including FLI-1, are thought to be responsible for causing Ewing's sarcoma. However, the ectopic expression of EWS/FLI-1 by itself is insufficient for carcinogenesis, suggesting that additional events are required. We have reported that the proline-rich Akt substrate of 40 kDa (PRAS40) is an EWS target gene and contributes to the carcinogenesis of Ewing's sarcoma. The expression of PRAS40 protein is increased in Ewing's sarcoma cells and the knockdown of PRAS40 significantly repressed the proliferation, migration and invasion of Ewing's sarcoma cells. The knockdown of PRAS40 was found to induce apoptosis in melanoma cells. The introduction of PRAS40 in mouse brain protects neuronal cells from apoptosis, and the phosphorylation of PRAS40 at Thr246 by Akt is proposed to be important for the prevention of apoptosis. Our previous data that the activated form of caspase 3, cleaved caspase 3, was markedly increased in the PRAS40-knockdown Ewing's sarcoma cells implicated the potential of PRAS40 repressing apoptosis. However, the mechanism remains largely unknown..Since the phosphorylation of Akt (pAkt) was decreased in PRAS40-knockdown cells in response to insulin treatment, we hypothesize that PRAS40 might regulate the activity of Akt through positive feedback, which results in suppressing apoptosis and promoting cell proliferation, migration and invasion. Here, to clarify the mechanism of the carcinogenetic role of PRAS40, we plan to investigate further the effect of PRAS40 on apoptosis of Ewing's sarcoma cells by Flow Cytometer, TUNEL and DNA ladder assay. Next we should investigate the expression or the activity of the factors upstream of Akt including PTEN, PI3K, PDK1 and growth factor receptor，and exclude the mechanism that PRAS40 regulates the activity of Akt. Simultaneously, we plan to screen the proteins interacting with PRAS40 in Ewing's sarcoma cells by proteomics approaches and explore the relationship between PRAS40 and the interacting factors relating to proliferation or apoptosis. Therefore, it is expected that in this project we would clarigy the mechanism that PRAS40 suppresses apoptosis, promotes the proliferation, migration and invasion of Ewing's sarcoma cells, and provide novel targets for the development of new treatment of Ewing's sarcoma.

尤文肉瘤是一种恶性度高，极易转移的肿瘤，具有染色体易位，导致EWS基因与ETS转录因子家族形成融合蛋白EWS/ETS。但是融合蛋白并不能独自引起尤文肉瘤形成。申请人发现在尤文肉瘤细胞中PRAS40蛋白表达增强，基因敲除PRAS40显著抑制细胞增殖，迁移和侵袭，同时caspase3活化型水平增高。因前期研究结果显示基因敲除PRAS40后Akt磷酸化水平降低，所以我们推测PRAS40在尤文肉瘤细胞中可能通过正反馈来参与Akt活性的调节，进而抑制凋亡，促进细胞增殖，迁移和侵袭。为弄清PRAS40在尤文肉瘤细胞中致癌作用的机制，本项目拟通过进一步调查基因敲除PRAS40对尤文肉瘤细胞的凋亡和Akt上游因子活性的影响，弄清PRAS40调节Akt活性的机理。并应用蛋白质组学的方法来全面筛选并确定在尤文肉瘤细胞中与PRAS40相互作用的因子，从而揭示PRAS40的致癌机制，有望为新药开发提供新靶点。

尤文肉瘤是一种恶性度高，极易转移的肿瘤，具有染色体易位，导致EWS基因与ETS 转录因子家族形成融合蛋白EWS/ETS。但是融合蛋白并不能独自引起尤文肉瘤形成。申请 人前期工作发现在尤文肉瘤细胞中PRAS40蛋白表达增强，siRNA干扰PRAS40显著抑制细胞增殖，同时caspase3活化型水平增高。我们推测PRAS40在尤文肉瘤细胞中可能通过调控凋亡，促进细胞增殖。.为进一步研究PRAS40在尤文肉瘤发生发展中的作用，并揭示其作用机制，我们通过TUNEL实验发现用siRNA干扰PRAS40表达后，两株尤文肉瘤细胞A673和TC-32均表现出TUNEL阳性细胞增多的趋势，细胞色素C的释放和caspase3活化型表达均升高，提示siRNA干扰PRAS40引起细胞凋亡。为进一步揭示PRAS40调控尤文肉瘤细胞凋亡的作用机制，我们检测了Akt和mTOR信号通路的活化情况，发现RNA干扰PRAS40导致Akt、和mTOR下游分子，S6和S6K的磷酸化水平下调，而过表达PRAS40后，其磷酸化水平均被上调。在siRNA干扰PRAS40表达的同时，过表达活化型Akt (Myr-Akt)，发现细胞凋亡并没有被完全逆转，而且S6的磷酸化水平同样没有恢复。这些结果提示PRAS40调控细胞凋亡的作用靶点应该在Akt和mTOR的上游。之后，我们发现siRNA干扰PRAS40导致胰岛素受体磷酸化受抑，而过表达PRAS40可促进其磷酸化。如此，PRAS40可直接作用于胰岛素受体并刺激其磷酸化，从而激活Akt和mTOR通路，异常调控尤文肉瘤细胞的凋亡。而且，我们在小鼠移植瘤的实验中得出了同样的结果，siRNA干扰PRAS40后的尤文肉瘤细胞在裸鼠体内成瘤性大大降低，并且免疫组化的结果提示胰岛素受体和S6的磷酸化明显受抑。以上研究结果提示PRAS40在尤文肉瘤中通过促进胰岛素受体磷酸化而异常活化Akt和mTOR通路，从而异常调控凋亡而促进肿瘤形成，有望成为尤文肉瘤治疗的新靶点。.为彻底揭示PRAS40在尤文肉瘤发生发展中的作用网络，我们通过免疫沉淀法将PRAS40沉淀，从而纯化与其相互作用的蛋白，并应用质谱进行鉴定，确定了在尤文肉瘤细胞中与PRAS40相互作用的因子，其中CTTN系肿瘤转移相关因子，PGK1系糖代谢关键酶，PRKA1RB系PKA的调控组件，与肿瘤形成有重要联系。上述PRAS40相