GEFT调控Rac1/Cdc42-mTOR信号通路影响横纹肌肉瘤细胞自噬和凋亡的分子机制研究

Rhabdomyosarcoma (RMS) is the most common childhood malignant soft tissue tumor. Even though through multimodal therapies, the prognosis of the high-risk RMS patients is not good, with a 5-year overall survival rate of20–30%. Mediating autophagy and promoting death of tumor cell have been a novel promising cancer therapeutic strategy. In previous studies, we have showed that over expression of GEFT was closely related with metastasis and prognosis. It could promote proliferation, and invasion of tumor cell via GTPases /Rac1/Cdc42. A new study suggests that Rac1/Cdc42-mTOR signaling pathway has close relation with autophagy and apoptosis. Therefore, we hypothesize that GEFT could regulate rhabdomyosarcoma cell autophagy to influence proliferation and apoptosis of tumor cell. To verify this hypothesis, we plan to firstly analyze the relationship between the expression of GEFT, Rac1/Cdc42 and mTOR and the condition of the autophagy and apoptosis in tumor tissues, respectively. Secondly, using knockdown of GEFT, Rac1 or Cdc42 expression by shRNA or small inhibitor and inducing overexpression of them, we will investigate its influence on the activity of autophagy and apoptosis of cancer cells. Lastly, we will illuminate GEFT-Rac1/Cdc42-mTOR signaling pathway regulating mechanism of autophagy and apoptosis in rhabdomyosarcoma by animal model. This project will find a novel target and direction for rhabdomyosarcoma therapy.

横纹肌肉瘤是儿童最常见软组织恶性肿瘤，尽管运用综合治疗，高风险患者5年生存率仅20-30%，而调控自噬水平促进肿瘤细胞死亡是肿瘤治疗的新策略。我们前期研究显示GEFT高表达与横纹肌肉瘤的转移、预后密切相关且通过GTPases/Rac1/Cdc42发挥促肿瘤细胞增殖、侵袭的作用，而最新研究报道表明Rac1/Cdc42也参与mTOR通路介导的肿瘤细胞自噬和凋亡。因此我们推测GEFT可能通过Rac1/Cdc42-mTOR通路调控横纹肌肉瘤细胞的自噬影响增殖和凋亡。为验证假说，本项目首先分析GEFT、Rac1/Cdc42、mTOR表达与肿瘤自噬和凋亡的关系；其次，通过诱导和阻断GEFT、Rac1/Cdc42、mTOR表达，研究细胞自噬活性和凋亡的改变，最后，构建动物模型阐明GEFT-Rac1/Cdc42-mTOR通路在横纹肌肉瘤自噬和凋亡中的作用机制，为横纹肌肉瘤治疗提供新靶点和方向。

横纹肌肉瘤是儿童最常见软组织恶性肿瘤，尽管运用综合治疗，高风险患者5年生存率仅20-30%，而调控自噬水平促进肿瘤细胞死亡是肿瘤治疗的新策略。我们前期研究显示GEFT高表达与横纹肌肉瘤的转移、预后密切相关且通过GTPases/Rac1/Cdc42发挥促肿瘤细胞增殖、侵袭的作用，而最新研究报道表明Rac1/Cdc42也参与mTOR通路介导的肿瘤细胞自噬和凋亡。本课题实验结果显示，与对照组相比，横纹肌肉瘤组织中Rac1、Cdc42、p-mTOR、Bcl-2蛋白高表达，Beclin1、LC3、Bax蛋白低表达且具有统计学意义（P ＜ 0.05）。过表达GEFT能够抑制横纹肌肉瘤细胞自噬和凋亡，加入Rac1、Cdc42、mTOR抑制剂后，与过表达GEFT组相比，能够促进自噬和凋亡, 且Rac1、Cdc42可以调控mTOR抑制自噬和凋亡（P ＜ 0.05）。裸鼠实验结果表明GEFT通过Rac1/Cdc42途径促进横纹肌肉瘤细胞的生长和转移, 过表达GEFT能够抑制横纹肌肉瘤组织中自噬和凋亡相关蛋白的表达，且在加入Rac1、Cdc42抑制剂后，与过表达GEFT组相比，能够促进自噬和凋亡相关蛋白的表达（P ＜ 0.05）。综上所述，体内外实验初步证实GEFT能够通过激活Rac1/Cdc42-mTOR信号通路抑制横纹肌肉瘤的自噬和凋亡，为横纹肌肉瘤的发病机制提供新的思路并为其治疗提供新的靶点。