胞内补体激活在结直肠癌发生发展中的作用及其机制研究

Colorectal cancer (CRC) is one of the most common malignant cancers of gastrointestinal tract. Based on epidemiological studies, the etiology of colorectal cancer is multifactorial and complicated, including inherited genetic disorders, environmental and food-borne mutagens, chronic intestinal inflammation and so on. So far, it has been reported that the complement system was extensively activated in patients with colorectal cancer. Nevertheless, the role of complement activation in CRC development is poorly understood. In the previous study, we have demonstrated that C5 and C5ar1-deficiency almost completely prevents AOM/DSS-induced mouse colorectal cancer (CRC) development and the local level of β-catenin in C5 and C5ar1-deficient mice was dramatically lower than those in WT mice, suggesting the critical role of C5a/C5aR1 signaling in CRC development. Furthermore, we also detected C5a, a C5 activation fragment, in a variety of colonic epithelial cells and its receptor C5aR1 located only in the cytoplasm, but not membrane. Herein, we propose that, C5 could be cleavaged by a specific protease in colonic epithelial cells and its active fragment C5a could stabilize β-catenin by binding to its receptor C5aR1 intracellularly. Once β-catenin was activated, the expression of its downstream target genes, such as Ccnd1 and Cox2 could be up-regulated, thus promoting the development of colorectal cancer. Overall, this project intends to verify the above hypothesis from the perspectives of molecular, cellular, animal model and clinical samples, and elaborate the mechanisms of intracellular C5 activation and β-catenin stabilization through C5a/C5aR1 in colonic epithelial cells, so as to provide a new insight into the prevention or treatment of colorectal cancer in the future.

结直肠癌是常见的消化道恶性肿瘤之一，其发病机制复杂，涉及遗传、环境、炎症等诸多因素。研究发现，补体系统在结直肠癌中广泛激活，但关于其发挥的作用及机制鲜有报道。我们前期发现敲低C5和C5ar1基因可显著抑制小鼠结肠炎癌转化进程，并降低肠道局部β-catenin的水平，提示C5a/C5aR1信号对结直肠癌发生至关重要。此外，我们还证实肠上皮细胞中C5显著激活，产生C5a，且其受体C5aR1只定位于胞质中。故我们推测：肠上皮细胞中C5经特异蛋白酶切割后，产生的活性片段C5a与胞内C5aR1结合，增强β-catenin的蛋白稳定性，β-catenin激活后可入核上调Cyclin-D1、COX-2等靶基因的表达，从而促进结直肠癌的发生发展。本项目拟从分子、细胞、动物模型和临床样本四个层面验证该假说，阐明肠上皮细胞中C5激活及其介导β-catenin稳定的作用与机制，为未来结直肠癌的防治开拓新的思路。

补体系统是机体固有免疫的重要组成部分，其不仅能在细胞外识别并清除外来病原体，而且在细胞内环境中亦发挥重要作用。免疫细胞内补体组分C3和C5的激活对其内稳态的维持和免疫功能的调控具有重要意义。然而，目前关于肿瘤细胞内是否存在补体激活及其作用尚不清楚。我们研究发现在肠癌细胞的溶酶体和内体中，组织蛋白酶D（cathepsin D，CTSD）可以特异性切割C5产生活性产物C5a。C5a与其胞内受体C5aR1结合后触发了β-catenin稳定复合体（C5a/C5aR1/KCTD5/cullin3/Roc-1/β-catenin）的组装。该复合体促进β-catenin发生K63连接多聚泛素化修饰，并抑制其K48连接多聚泛素化修饰，从而抑制其蛋白酶体降解、增强蛋白稳定性并诱导其发生核易位激活下游信号。此外，C5或C5aR1基因缺失或使用C5aR1拮抗剂PMX205处理均可通过影响β-catenin的蛋白稳定性进而显著抑制小鼠肠炎相关性肠癌及自发肠道肿瘤的发生。在人结直肠癌（colorectal cancer，CRC）临床样本中，β-catenin表达水平增高与C5aR1，C5a及CTSD的高表达和不良预后密切相关。更重要的是，细胞内C5a/C5aR1信号介导的β-catenin的蛋白稳定也普遍存在于其他类型的细胞中。因此，本研究揭示了一种新的由胞内C5a/C5aR1信号介导的β-catenin稳定机制，其能促进肿瘤的发生发展，也提示了靶向胞内C5a/C5aR1信号可能是一种具有潜在应用价值的肿瘤预防和治疗策略。

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