E2F在肾透明细胞癌转移中的功能及其与VHL关系的研究

Progonosis of ccRCC is determined by tumor metastasis. VHL-HIF hypoxia pathway was thought to be the main mechanism of ccRCC metastasis, but only 60% ccRCC patients were found having VHL mutation, and therapeutic effect targeting VEGF was not so good. Preliminary study show that genes like FOXO3a, Klf4,S100A6 were highly related with ccRCC metastasis, and signal-net analysis show that they were closed related with E2F. As well as that, E2F1 had a high expression level in both VHL (-) and VHL (+) ccRCC, and it was related with invasiveness of ccRCC. So we hypothesized that E2F can affect metastasis of ccRCC by influence expression of FOXO3a in a way independent of VHL. This study plans to study the mechanism of E2F in promoting metastasis of ccRCC and its correlation between VHL through vitro an in vivo experiments, and then , do correlation analysis between E2F expression level and ccRCC clinical features. Key point of this study is aim to discuss whether E2F plays pivotal role in metastasis of ccRCC in a non VHL-HIF pathway, and to analyze weather E2F can be used as a therapeutic target, which can make up for the inadequacy of current treatments.

ccRCC的转移决定预后，VHL-HIF通路被认为是ccRCC转移的主要机制，但只有约60%的患者存在此通路的改变，而且针对下游VEGF的靶向治疗效果不理想。前期对ccRCC转移机制的研究发现，FOXO3a、KLF4、S100A6等基因与ccRCC的转移密切相关，这些基因的signal-net分析结果显示它们与E2F高度相关。其中E2F1在VHL（-）、VHL(+)的ccRCC中均异常高表达，并与ccRCC的侵袭力相关。因此我们假设E2F不依赖于VHL、在 ccRCC中通过影响FOXO3a等基因的表达而影响肿瘤的转移。本课题拟通过体外干扰、体内示踪等实验探讨E2F在ccRCC转移过程中的作用机制及其与VHL的关系；结合ccRCC肿瘤组织标本分析E2F与ccRCC预后等临床特征的相关性。重点探讨E2F是否在ccRCC转移过程中通过非VHL-HIF通路起关键作用，能否作为ccRCC的治疗靶点。

本项目结合ccRCC肿瘤组织标本分析E2F与ccRCC预后等临床特征的相关性，通过肿瘤样本免疫组化染色实验检测，发现E2F1在肿瘤中显著高表达，并且在转移性样本中表达量更高，其高表达的患者总体生存更差；二是采用细胞模型，结合干扰技术，在细胞水平对E2F1蛋白的功能进行了系统评价，包括对增殖，迁移和侵袭能力的影响，进一步采用裸鼠移植瘤模型，在体内证明了E2F1发挥了非常重要的促增殖转移功能。并找到了调控E2F1表达的重要转录因子KLF6，证实了KLF6与E2F1的表达呈负相关，并且也通过细胞和小鼠模型，验证了KLF6在ccRCC增殖转移中发挥了重要的调控功能。. 因此，E2F1与KLF6有可能成为肾透明细胞癌预后评估和新的治疗靶点，为临床治疗寻找新的途径和线索。