KLF4表达缺失活化肝癌TGF-β/Smads信号通路的作用及机制研究

Overactivation of TGF-β signaling pathway plays an important role in hepatocellular carcinoma (HCC) progression; however the underlying mechanism remains unknown. Our previous data published in Clinical Cancer Research revealed that loss of the tumor suppressor Krüpple-like factor 4 (KLF4) expression in human HCC significantly correlated with dedifferentiation, invasion and metastasis of the tumor. We further discovered that loss of KLF4 expression closely associated with phosphorylation of Smad2/3, the key signal mediators of canonical TGF-β signaling pathway, using tissue microarray analyses. Moreover, KLF4 overexpression in HCC cells inhibits nuclear translocation andphosphorylation of Smad2/3, whereas deletion of KLF4 Zinc-finger domain (ZFD) abrogated this effect. On the basis of these findings, we propose that KLF4 inhibits TGF-β signal transduction at the transcriptional level, and thus loss of KLF4 expression leads to overactivation of TGF-β pathway. In this project, the impact of KLF4 on TGF-β/Smads pathway and cell behavior will be investigated using transfected cells. The molecular mechanism regarding the regulatory effect of KLF4 on TGF-β pathway will be explored by construction of promoter reporters conjugated with dual luciferase assay. In vivo study will be conducted by establishment HCC mouse models in liver-conditioned Klf4 knockout and knockin mice. This project will extend our knowledge on activation of TGF-β pathway and the tumor suppressor KLF4 in HCC progression, and may provide important theoretical basis for future TGF-β targeted therapy.

TGF-β/Smads通路过度活化是肝癌进展的重要原因，其产生机制不明。课题组前期在«Clin Cancer Res»杂志报道肝癌KLF4表达缺失，与肝癌去分化及复发相关，进一步研究发现KLF4缺失与TGF-β信号枢纽分子Smad2/3磷酸化有关，KLF4过表达抑制Smad2/3磷酸化及入核，而去除KLF4 DNA结合域后该作用消失。由此推测KLF4通过转录作用抑制肝癌TGF-β/Smads信号转导，当KLF4缺失时TGF-β通路失控活化。本项目拟采用基因转染等方法，体外研究KLF4对肝癌TGF-β通路及细胞行为的影响；用双荧光素酶报告基因、ChIP等技术，探寻KLF4调控肝癌TGF-β/Smads通路的分子机制；建立肝脏Klf4条件敲除和敲入小鼠肝癌模型并综合临床样本进行体内研究。这将深化对肝癌TGF-β通路活化机制的理解，拓展对KLF4的认识，为TGF-β靶向治疗提供依据。

TGF-β/Smads通路过度活化是肝癌进展的重要原因。本研究发现抑癌转录因子KLF4表达缺失与TGF-β信号传导枢纽分子Smad2/3磷酸化有关，KLF4过表达抑制Smad2/3磷酸化及入核，而去除KLF4 DNA结合域后该作用消失，通过双荧光素酶报告基因检测进一步证实KLF4通过转录活性抑制p-Smad2/3的转录作用，而当KLF4缺失时TGF-β通路失控活化。采用基因转染等方法，体外细胞学研究证实KLF4抑制TGF-β1诱导的肝癌细胞侵袭迁移能力，并且该作用依赖其转录活性。机制研究进一步发现KLF4抑制Smad2/3的磷酸化与KLF4和Smad2/3的结合没有关系，而是通过KLF4转录调节TGF-β/Smads信号通路的抑制因子Smad7表达来实现的。我们构建Smad7启动子及突变体载体，荧光素酶报告基因检测联合ChIP显示KLF4直接结合Smad7启动子序列上KLF4的#2结合位点发挥转录激活作用。我们构建人肝癌组织芯片阵列，通过免疫组化发现人肝癌组织中KLF4表达缺失，KLF4表达水平与Smad7蛋白表达正相关，与p-Smad2/3呈负相关，KLF4、Smad7低表达，p-Smad2、p-Smad3高表达与肝癌术后不良预后相关，KLF4低表达是独立危险因素。因此，我们得出KLF4通过直接结合Smad7启动子序列上的KLF4结合位点，正向调节Smad7转录，促使Smad7蛋白表达上调，进而抑制TGF-β/Smads信号通路，而在人肝癌组织中KLF4表达缺失致使Smad7表达减少，从而使得TGF-β/Smads信号通路抑制因素减除导致其过度活，最终促进肝癌恶性进展。在上述研究基础上，课题组还对肝癌内KLF4表达缺失原因进行机制上的初步探索，发现TGF-β/Smads-FAT10-KLF4信号轴，TGF-β/Smads信号活化通过上调FAT10导致KLF4降解致使其表达缺失，形成TGF-β/Smads信号活化与KLF4表达缺失的恶性循环。本研究深化了对肝癌TGF-β通路活化机制的理解，拓展了对KLF4的认识，可为肝癌靶向TGF-β治疗及患者预后判断提供重要依据。