PDCD4-Akt反馈环路在胃癌顺铂耐药中的作用机制研究

Cisplatin (DDP) resistance is a major driver of therapeutic failure in gastric cancer. Previous research found that Akt play an important role in cancer multidrug resistance. programmed cell death 4 (PDCD4), a recently identified tumor suppress gene, plays a critical role in chemotherapeutic resistance and poor prognosis of several cancer types. Previous studies show that Akt plays a key role in multiple durg resistance through YB-1. However, the role of PDCD4 and Akt involved in the pathogenesis of DDP resistance have not been addressed. In our previous study, PDCD4 has lower expression in DDP resistant cell lines than in DDP sensitive cell lines and correlates with levels of pAkt; overexpression or silence of PDCD4 caused reversed expression level of pAkt and related with sensitivity to DDP. Therefore, we focus on the function and mechanism of PDCD4-Akt feedback pathway in regulating DDP resistance of gastric cancer. This study includes: 1. We investigate the expression of PDCD4 and correlation between pAkt expression and chemotherapy sensitivity and prognosis; 2. We construct cell model with higher or lower PDCD4 expression to detect the function and molecular mechanism of PDCD4/Akt/YB-1 pathway in DDP resistance; 3. We verify the function and molecular mechanism of PDCD4-Akt regulate DDP resistance in vivo mice model. Our study will explain the role of PDCD4-Akt in DDP resistance and show that PDCD4 may be an important maker for human gastric cancer chemotherapy sensitivity as well as a potential therapeutic target.

顺铂(Cisplatin, DDP)是顺铂是目前临床上使用的最重要的抗癌药物之一。但是，肿瘤原发或继发性耐药的产生是导致临床胃癌化疗失败的主要原因。研究发现，Akt可以通过YB-1参与肿瘤多药耐药。细胞程序性死亡基因4 (programmed cell death 4, PDCD4)是近年来新发现的一种抑癌基因。而我们前期的研究发现，DDP耐药的人胃癌细胞胞系中PDCD4的表达量显著下降；PDCD4的表达能够反向调控pAkt的水平，并且与DDP敏感性相关。本项目拟首先通过临床样本，分析PDCD4/Akt活化与患者病理及化疗反应的相关性；通过构建PDCD4高表达和低表达的SGC7901细胞株，研究PDCD4/Akt/YB-1在胃癌DDP耐药中的作用的分子机制；用动物模型验证PDCD4及Akt在体内的耐药作用。本研究如获成功，PDCD4有望成为判断胃癌DDP敏感性的指标和DDP耐药治疗的靶点。

细胞程序性死亡基因4（programmed cell death 4, PDCD4）是近年来新发现的一种抑癌基因。多项研究分别证实PDCD4可以调节多种肿瘤细胞对顺铂的敏感性。pAKT作为一个与化疗耐受相关的关键信号分子，能活化多药耐药相关基因的表达。近期研究发现，pAKT能够介导PDCD4的磷酸化发生，从而促进PDCD4蛋白的降解。但是PDCD4是否能够反向调控pAKT通路参与胃癌顺铂耐药尚未见报道。本项目主要通过免疫组织化学﹑MTT﹑克隆形成实验﹑Western-blot﹑流式细胞术﹑双荧光素酶报告基因实验﹑染色质免疫共沉淀和裸鼠致瘤实验等方法研究PDCD4影响胃癌顺铂耐药的主要分子机制。主要研究包括：1. PDCD4的表达与胃癌细胞顺铂耐药的相关性；2.在胃癌顺铂敏感细胞系中构建稳定敲低表达PDCD4的细胞株，检测PDCD4表达缺失对pAKT/pYB1/MDR1通路的影响。3. 在胃癌顺铂耐药细胞系中构建稳定过表达PDCD4的细胞株，检测PDCD4过表达对pAKT/pYB1/MDR1通路的影响。4. 染色质免疫共沉淀检测PDCD4-YB1-MDR1的启动子区域的结合作用。5. 裸鼠移植瘤实验证实体内PDCD4与顺铂治疗的相关性。而通过本项目的研究发现，PDCD4的表达水平与胃癌细胞对顺铂的耐药性密切相关，PDCD4能够负向调控pAKT/pYB1/MDR1通路的表达。进一步的研究显示PDCD4能够通过直接结合YB1蛋白，抑制YB1磷酸化入核结合MDR1的启动子区域，从而发挥其对MDR1的转录抑制作用。因此，我们推断PDCD4与pAKT构成的反馈环路在胃癌顺铂的耐药中发挥重要作用。PDCD4可能作为评价胃癌顺铂敏感性和预后的分子靶标，为临床化疗提供理论依据。