CREB5促进KRAS突变型结直肠癌侵袭转移的作用机制及其潜在临床应用价值研究

Targeting therapy in KRAS-mutated colorectal cancer is one of biggest problem in prevention and treatment of metastatic colorectal cancer currently. Our preliminary data demonstrated that CREB5 is one of the key points in the metastatic-associated signaling network of colorectal cancer. In addition, CREB5 might be involved in the downstream signaling of oncogenic KRAS. However, the role and underlying mechanism of CREB5 in the progression of human cancers remains unknown. In the present study, we will apply the following researches, based on our previous study. First, we will establish stable CREB5-overexpressing and shRNA knocking down cell lines, and further establish the CREB5-knockout cell lines through CRISPR/Cas9 method. Then, we will establish orthotopic colon metastatic mouse models using these cell lines. Based on the cell lines and orthotopic colon metastatic mouse models, we will observe the role of CREB5 deregulation in proliferation, tumorigenesis, invasiveness, as well as metastasis, both in vitro and in vivo. Moreover, we will analyze the underlying mechanism and regulating network through which CREB5 promote progression of colorectal cancer, through Co-IP, CHIP-seq, RNA-seq, gene expression microarray, as well as bioinformatics analysis. Furthermore, we will analyze the association between KRAS mutation and CREB5 expression in a large cohort of colorectal cancer tissues. We will further investigate whether inhibiting CREB5 can alter the efficiency of the clinically applied targeting therapy drugs. The results of this project will provide a comprehensive understanding of the role and mechanism of CREB5 deregulation in invasion and metastasis of colorectal cancer, and provide new evidence for developing novel strategies in targeting KRAS-mutated colorectal cancer.

针对KRAS突变型结直肠癌的靶向治疗是目前防治转移性结直肠癌的重点和难点之一。我们的前期研究提示CREB5基因是结直肠癌转移相关信号通路网络中的关键调控点，并可能参与突变型KRAS下游信号通路，然而CREB5在肿瘤侵袭转移中的作用机制不清楚。本项目将在前期研究基础上，建立CREB5过表达、干扰及CRISPR/Cas9法敲除CREB5基因的结直肠癌细胞株、可视化结肠癌原位移植转移动物及肠道特异性CREB5转基因鼠模型；分析CREB5对结直肠癌细胞增殖成瘤及侵袭转移能力的影响；采用CoIP、CHIP-seq、RNA-seq、表达谱芯片等方法，结合生物信息学，分析CREB5在结直肠癌演进过程中的作用机制和调控网络；在组织标本中验证KRAS突变与CREB5的相关性；并通过体内外实验，观察干预CREB5后，KRAS突变型结直肠癌细胞对靶向治疗药物的敏感性，为转移性结直肠癌的治疗和药物筛选提供新思路。

研究背景：cAMP反应元件结合蛋白5（CREB5）是真核细胞中的一种转录因子，参与调节基因的表达。前期研究中，我们使用生物信息学分析发现CREB5参与了结直肠癌（CRC）的发生和发展。但目前对于CREB5在CRC中的生物学作用和潜在的调控机制尚不清楚。.研究方法：我们使用实时荧光PCR，蛋白质印迹和免疫组化检测了CREB5的表达。在体外，我们使用了肿瘤细胞迁移实验、划痕实验，鸡绒毛尿囊膜试验和脉管形成实验检测了 CREB5对CRC细胞的迁移和肿瘤血管生成能力的影响。另外，为了验证CREB5在体内的作用，我们在裸鼠中进行了肿瘤的原位植入实验。同时，我们使用了基因集富集分析（GSEA分析）来探索CREB5在CRC中的潜在机制。.研究结果：我们发现CREB5在结直肠癌中的表达显著上调。其高表达与CRC患者的WHO分期晚期和TNM分期以及生存期短呈正相关。此外，CREB5的过表达促进了体外和体内CRC细胞的侵袭性和转移能力，而CREB5的沉默与之相反。机制上，我们发现CREB5直接与MET启动子区结合并激活肝细胞生长因子-MET信号通路。更为重要的是，MET的抑制减少了CREB5过表达的CRC细胞的侵袭和转移，提示CREB5主要通过激活MET信号来促进转移。.结论：我们的研究证明了CREB5在CRC转移中的关键作用，其机制主要是直接上调MET表达。因此，CREB5可能是有效克服CRC转移的潜在预后标志物和治疗靶标。