CHD4/NuRD抑制FOXE3表达及其在促进肝癌转移中的作用机制

Recurrence and metastasis remain the most common causes of lethal outcomes in hepatocellular carcinoma (HCC) after curative resection. Thus it is critical to discover the mechanisms underlying HCC metastasis. Forkhead box (Fox) proteins are a family of evolutionarily conserved transcriptional regulators that play important roles in both normal biological processes and in cancer development. Previously, we discovered that: ①FOXE3 expression was markedly lower in HCC tissues than in adjacent noncancerous tissues and normal liver tissues. HCC patients with low expression of FOXE3 had shorter overall survival and higher recurrence rates than those with high expression of FOXE3. ②Knockdown of FOXE3 significantly increased the migration, invasion, and lung metastasis of HCC cells, whereas over-expression of FOXE3 decreased these processes. ③CHD4/NuRD complex inhibited FOXE3 transcription. Based on these studies, we hypothesize that CHD4/NuRD directly binds to the promoter of FOXE3 and inhibits its expression. FOXE3 inhibition reactivates FOXE3 target genes’ expression, thereby leading to the invasion and metastasis of HCC. However, what is the molecular mechanism of FOXE3-mediated HCC metastasis? How CHD4/NuRD complex inhibits FOXE3 transcription? Does inhibition of CHD4/NuRD-FOXE3 pathway decrease HCC metastasis? In this project, we are going to answer these important questions. We are going to use cell lines, nude mouse model, conditional knockout mouse model, and human HCC samples to study these questions. These studies not only discover the new function of FOXE3, but also provide potential a candidate biomarker for HCC prognosis and a target for new therapies.

转移是导致肝癌死亡率高的主要原因，FOX转录因子在其中发挥重要调节作用，然而分子机制不明。我们率先发现：①FOXE3在肝癌中表达显著低于癌旁和正常肝组织，FOXE3低表达患者复发率高生存率低，与肝癌预后不良密切相关；②下调FOXE3促进肝癌侵袭和转移，上调FOXE3抑制肝癌侵袭和转移；③CHD4/NuRD复合物抑制FOXE3转录。据此推测：CHD4/NuRD通过与FOXE3启动子结合抑制其表达，被FOXE3转录抑制的直接调控靶基因由此解除抑制导致表达上调，从而促进肝癌侵袭和转移。然而，FOXE3抑制肝癌转移的分子机制是什么？CHD4/NuRD如何抑制FOXE3表达？干预CHD4/NuRD-FOXE3途径能否有效抑制肝癌转移？本课题拟通过细胞、裸鼠、条件敲除鼠、临床组织标本等一系列实验阐明以上三个问题。研究结果不仅有望拓展对FOXE3功能的新认识，而且为肝癌治疗提供新的切入点和思路。

转移是导致肿瘤死亡率高的主要原因，因此探讨肿瘤转移的分子机制至关重要。肝癌转移过程中涉及大量基因表达上调和基因表达沉默，转录因子通过结合到基因启动子区域在转录水平激活或抑制基因表达，在这其中发挥着关键的调控作用。FOX基因家族是一类是在进化上高度保守，参与胚胎及器官发育以及干细胞自我更新和分化的重要转录因子。该家族作为调节细胞命运的重要基因，一旦表达失常将改变细胞命运甚至癌变。在本项目标书内容的基础上拓展和延伸，解析了以FOXE3, FOXC1, FOXK2转录因子为关键节点的上下游信号调控网络在促进肿瘤转移中的不同作用和机制。关键发现如下：（1）肝癌组织中FOXE3的低表达与脉管侵袭、TNM分期、远处转移正相关，FOXE3低表达组患者复发率高生存率低，FOXE3低表达可以作为判断肝癌预后不良的独立预测因素。肝脏原位种植模型发现下调FOXE3表达显著增强肝癌细胞的迁移、侵袭和转移能力，而上调FOXE3表达显著抑制肝癌细胞的迁移、侵袭和转移能力。CHD4-NuRD转录抑制复合物结合到FOXE3基因启动子区域抑制其表达，FOXE3表达缺失从而促进肝癌转移。（2）活性氧激活ERK1/2信号，促进ELK1结合到FOXC1基因启动子上转录激活FOXC1表达，FOXC1上调DNA甲基化酶DNMT3B表达，导致CTH基因启动子被甲基化，从而导致CTH基因表达被抑制。CTH是半胱氨酸代谢重要的调节分子，CTH表达被抑制后，引起细胞内更多的ROS聚集，从而促进肝癌转移。（3）表皮生长因子EGF通过EGFR/ERK/NF-κB途径激活FOXK2；一方面，FOXK2转录激活E盒锌指结合蛋白ZEB1表达显著增强肿瘤细胞侵袭和运动能力；另一方面，FOXK2转录激活EGF的特异性受体EGFR表达，形成EGF/FOXK2/EGFR正反馈信号途径导致该信号通路持续激活，从而促进肿瘤细胞发生肝转移和远处肺转移。