奥利司他(orlistat)抑制Wnt信号通路和大肠癌的分子机制研究

Colorectal cancer is a type of disease with high morbidity and mortality in the world, it is urgent to develop effective therapeutic agents for clinical use. Large-scale genomic sequencing has revealed that oncogenic mutation of Wnt/β-catenin signaling pathway is a key causing driver of colorectal cancer, and inhibitor of Wnt signaling is believed to benefit colorectal cancer patients. Recently we have discovered orlistat, a FDA approved drug designed to treat obesity, significantly inhibited Wnt/β-catenin signaling in various in vitro and in vivo models. Importantly, orlistat inhibited the clonegentic growth of colorectal cancer cells and reduced the intestinal adenomas in Apc mutant mice with little toxicity. We also profiled the direct binding protein of orlistat using chemical proteomics approaches. Based on the preliminary data, the project was designed to elucidate the underlying action mechanisms of orlistat. We will firstly identify the functional target proteins of orlistat, then ask how orlistat bind to and modulate the target proteins, finally we will figure out the mechanism for the target protein regulating Wnt/β-catenin signaling and colorectal cancer. The project is expected to provide molecule evidence supporting the potential benefit of orlistat to colorectal cancer patients, but also provide a full picture of orlistat target proteins and new indications.

大肠癌是发病率和死亡率最高的肿瘤之一，急需开发新型治疗药物。大规模基因组测序表明Wnt/β-catenin信号通路是大肠癌最重要的治疗靶标。前期工作中，申请人在多个模型中证实了减肥药奥利司他(orlistat)是个新型Wnt信号通路抑制剂和大肠癌治疗药物，并且利用化学蛋白组学技术高通量鉴定了orlistat直接结合蛋白，但其分子机制尚不清楚。在此基础上,本项目聚焦于orlistat抑制Wnt通路和大肠癌的分子机制研究，首先鉴定orlistat抑制Wnt通路的功能靶标蛋白，然后阐明orlistat结合和调控靶标蛋白的机制，最后探讨orlistat靶标蛋白调控Wnt通路和大肠癌的分子机制。本项目不仅可以为orlistat应用于大肠癌临床治疗奠定理论基础，并且有望阐明orlistat新靶标蛋白和新适应症，具有重要的科学意义和社会价值。

大肠癌是发病率和死亡率最高的肿瘤之一，急需开发新型治疗药物。大规模基因组测序表明Wnt/β-catenin信号通路是大肠癌最重要的治疗靶标。本项目在多个模型中证实了减肥药奥利司他(orlistat)是个新型Wnt信号通路抑制剂和潜在的大肠癌治疗药物，但在结肠癌细胞中利用基因敲除技术证实该表型与已知靶标脂肪酸合成酶（Fatty acid synthase）无关。在此基础上, 本项目通过DARTS联用质谱技术，鉴定了NEDD8结合酶UBC12是orlistat在结肠癌的潜在靶标蛋白，并通过CETSA、MST和ITC等多种技术验证了两者在体内和体外实验中的直接结合。由于NEDD8是一种重要的类泛素，本项目进一步证实了NEDD8修饰不仅是β-catenin蛋白降解所必需，也与其转录活性高度相关。由于UBC12是目前已知的2个NEDD8结合酶中的一个，本项目确认了orlistat显著调控UBC12结合NEDD8的活性，及其底物cullin蛋白的NEDD8修饰水平，同时证实了UBC12与Wnt/β-catenin信号通路和结肠癌直接相关，并利用功能缺失实验证实UBC12是orlistat抑制Wnt/β-catenin信号通路所必需。值得注意的是，尽管UBC12是已知的肿瘤治疗靶标，但迄今为止尚未有直接靶向结合UBC12的小分子报道，本项目不仅鉴定了orlistat新靶标蛋白和新适应症，而且鉴定首个UBC12的靶向小分子，并证实UBC12对Wnt/β-catenin信号通路的调控作用，为基于UBC12和Wnt/β-catenin信号通路开发新型靶向药物提供了全新的先导分子，具有重要的科学意义和社会价值。