结直肠癌细胞中p53负调控ChREBP的机制和功能研究

Metabolic regulation in initiation and progression of colorectal cancer has been the subject of much biomedical research over the past several years. People are paying more and more attention to the new role of p53 in regulating metabolism and proliferation by modulating the level and activity of metabolic proteins in colorectal cancer cells. Our published and unpublished data show that downregulation of ChREBP, the transcription factor critical for metabolic regulation, increases p53 activity, induces cell cycle arrest and decreases proliferation in colorectal cancer cells. Moreover, the level and activity of ChREBP protein increases in p53 -/- colorectal cancer cells compared with p53 +/+ cells. p53 not only decreases ChREBP transcription, but also interacts with ChREBP protein and promotes ChREBP protein degradation in colorectal cancer cells. However, the mechanism underlying the negative regulation of ChREBP by p53 remains unclear. We aim to study, 1) the mechanism by which p53 inhibits ChREBP transcription; 2) the mechanism by which p53 interacts with ChREBP; 3) the mechanism by which p53 promotes degradation of p53 protein; 4) physiological function of the negative regulation of ChREBP by p53 in metabolism as well as initiation and progression of colorectal cancer using p53 and ChREBP knockout mice. Our project will provide clues for designing drugs for colorectal cancer prevention and treatment by regulating expression and activity of p53 and ChREBP.

近年来从代谢角度研究调控结直肠癌发生发展的新机制是该领域的一个热点，p53通过调控多种代谢相关蛋白表达和活性进而影响结直肠癌细胞代谢和增殖的新功能日益受到关注。我们的研究表明结直肠癌细胞中调控代谢的转录因子ChREBP表达下调可以增加p53活性、诱导细胞周期阻滞和抑制细胞增殖；p53缺失的结直肠癌细胞中ChREBP表达和活性增加；过量表达p53抑制ChREBP基因转录；p53与ChREBP蛋白相互作用并降低ChREBP蛋白稳定性。但是，p53负调控ChREBP的分子机制和生理功能不明。我们将研究：1）p53抑制ChREBP转录的机制；2）p53与ChREBP相互作用的机制；3）p53促进ChREBP蛋白降解的机制；4）运用敲除小鼠模型研究p53负调控ChREBP对于癌细胞代谢和结直肠癌发生发展的作用。本项目将为从调控p53和ChREBP表达及活性角度设计结直肠癌防治药物提供理论基础。

我们前期研究发现，在结直肠癌细胞系中，转录因子ChREBP与p53互为负调控因子，但此负调控的机制和功能不明。.在该项目的资助下，我们发现葡萄糖通过增加转录因子HNF-4α的水平和活性激活ChREBP基因的转录，p53可能通过降低HNF-4α的水平和活性抑制ChREBP转录。结直肠癌细胞中，E3连接酶Smurf2促进ChREBP蛋白的泛素化修饰和蛋白酶体途径降解。与对照相比，ChREBP敲除鼠AOM/DSS诱导的结直肠癌发生发展明显减缓。但是，ChREBP缺失没有改变肠道细胞p53基因表达和p53信号通路活性。与对照组相比，ChREBP敲除鼠肠道上皮细胞增殖明显降低。转录组学分析发现与增殖相关的Wnt/β-catenin信号通路多个靶基因在敲除鼠中下调。ChREBP可与β-catenin直接结合，促进β-catenin进入细胞核，增加Wnt下游靶基因的表达从而促进结直肠癌发生发展。并且，ChREBP通过其靶基因SCD-1改变饱和与不饱和脂肪酸比例，进而降低PTEN活性，促进结直肠癌细胞转移。.此外，我们发现ChREBP和p53互为负调控因子的现象虽然在结直肠癌中不显著，但是在肝损伤和肝癌细胞中非常明显。因此，我们在进展报告中指出部分研究内容的调整，即我们运用敲除鼠模型研究ChREBP负调控p53对于肝细胞DNA损伤和肝癌发生发展的作用。我们分别对野生和ChREBP敲除小鼠腹腔注射致癌剂二乙基亚硝胺（DEN）诱发急性肝损伤，与对照相比，ChREBP敲除鼠肝脏DNA损伤减轻。ChREBP缺失可减缓由DEN或DEN联合高脂饮食诱导的小鼠肝癌发生发展。在急性肝损伤和肝癌模型中，ChREBP缺失明显增加p53水平和p53下游靶基因如p21和XPC的表达。我们构建了ChREBP/p53双敲除小鼠品系，发现p53缺失可以增加DEN诱导的ChREBP敲除鼠DNA 损伤水平，说明p53在ChREBP调控肝细胞DNA损伤中发挥重要作用。.综上所述，我们不仅发现结直肠癌细胞中调控ChREBP水平和活性的新机制，找到ChREBP调控β-catenin细胞内定位与转录活性，增强Wnt信号通路活性和促进结直肠癌发生发展的新功能；而且首次发现ChREBP缺失通过增加p53信号通路活性减少肝细胞DNA损伤的重要作用。我们的研究可能为消化道肿瘤防治提供潜在的新靶点。