DJ-1及其氧化还原修饰参与结直肠癌发生发展的机制与临床意义研究

Colorectal cancer (CRC) is a common malignancy which causes global health burden. In our previous proteomics study, we identified DJ-1 as a redox-sensitive protein that harbors the potential to be candidate biomarker in reactive oxygen species (ROS)-induced migration and invasion in CRC cells (Lei et al. Molecular & Cellular Proteomics 2011). DJ-1 is involved in the regulation of cellular redox signaling cascades such as Akt signaling, and plays crucial roles in the defense against oxidative stress and cell death. However, the role that DJ-1 playing in the development and progression of CRC as well as the mechanisms regarding redox modification of DJ-1 remains unknown. Our further functional analysis revealed that: 1) the expression of DJ-1 was markedly up-regulated in tumor tissues compared with normal control, and was significantly correlated with the invasion depth, TNM stage and histo-differentiation; 2) the suppression of DJ-1 expression reduced the proliferative and migratory capacity of CRC cells; 3) the redox modification of DJ-1 was associated with metastatic potential of CRC; 4) interaction proteomics analysis implied the involvement of multiple potential DJ-1-binding proteins in various signaling pathways such as PI3K-Akt signaling. These results suggest that the expression and redox modification of DJ-1 play an important role in the development and progression of CRC. In this project, we will apply the molecular and cell biology strategies combined with animal models to comprehensively interrogate the biological functions of DJ-1 and clarify the role of its redox modification in the proliferation and metastasis of CRC cells. Further, we will use larger selected clinical cohorts to analyze the clinical and pathological significance of DJ-1. Our study aims to evaluate the potential of DJ-1 as an early diagnostic biomarker or drug target in CRC, which will pave the road for translational medicine.

结直肠癌是临床上常见的恶性肿瘤，严重危害人类身体健康。DJ-1是我们前期在活性氧促进结直肠癌转移研究中筛选到的标志物之一，结果发表于Mol Cell Proteomics杂志。该蛋白在抵御氧化应激和细胞死亡、以及调控Akt等多个关键通路方面均具有重要作用，但其在结直肠癌发生发展中的作用与机制仍不清楚。我们后续研究发现：DJ-1在结直肠癌中高表达，且与肿瘤浸润深度及分期分级等密切关联；干扰其表达可显著抑制结直肠癌细胞增殖和迁移能力；其氧化还原修饰与结直肠癌细胞的转移潜能相关；蛋白质相互作用分析结果也提示其可激活Akt等通路。本项目拟在此基础上，综合采用多种分子肿瘤学研究技术，深入研究 DJ-1异常表达以及ROS介导的氧化还原修饰在结直肠癌发生发展中的生物学功能，阐明DJ-1及其氧化还原修饰在结直肠癌发生发展中的作用机制与临床意义，探讨其作为结直肠癌早期诊断标志物或药物靶标的潜在应用价值。

我们在HCT116、SW480和SW620细胞里面构建了DJ-1稳定敲除或稳定过表达细胞，发现DJ-1可以促进结直肠癌细胞的增殖和迁移。在裸鼠体内建立了皮下瘤和肺转移模型，发现DJ-1可以在体内促进结直肠细胞的生长和转移。利用转录组测序结合分子机制实验我们证实DJ-1可以显著激活Wnt和Hedgehog 信号通路。DJ-1可以通过促进PLAGL2的表达来激活WNT信号通路介导BMP4的过表达来促进结直肠癌细胞的迁移、通过促进CCND1等WNT信号靶基因来促进结直肠癌细胞的增殖。Hedgehog信号通路也参与了DJ-1对BMP4表达和结直肠细胞增殖的调控。通过生物信息学分析TGGA数据库，利用Origene购买的结直肠癌cDNA芯片和从四川省人民医院收集的181例结直肠癌样本综合分析发现DJ-1在结直肠癌中高表达，并且与肿瘤的浸润深度、大小、分期、分级和预后相关。证实DJ-1可以作为结直肠癌的诊断标志物。我们发现潜在抗肿瘤药物环吡酮胺可以通过抑制DJ-1的表达来杀伤结直肠癌细胞，它降低DJ-1表达以后可以显著促进线粒体活性氧的产生，从而促进结直肠癌细胞死亡。DJ-1过表达会抑制环吡酮胺对结直肠癌的杀伤作用，证实DJ-1可以作为结直肠癌潜在的治疗靶点。我们检测了DJ-1稳定敲除或稳定过表达结直肠癌细胞内活性氧的积累情况，发现差异不大。我们进一步利用C53A，C46A和C106A DJ-1突变质粒在结直肠癌细胞中构建稳定细胞株，发现非应激水平，DJ-1的过表达和敲出并不会对细胞内ROS的积累产生太大的影响，DJ-1的三个主要半胱氨酸的突变仍然可以促进结直肠癌细胞的增殖和迁移，提示DJ-1的氧化还原调控能力并不是DJ-1在非应激环境下促进结直肠癌进展的主要原因。