5-FU耐药是结肠癌治疗过程中的一个常见的临床难题，而肿瘤细胞耐药多与凋亡途径的受阻有关。申请人之前的研究结果发现，特异性激活DR5凋亡路径可以更为有效地杀死结肠癌细胞，同时配合使用5-FU可以显著地增强治疗效果，其机制是5-FU处理后，DR5能以不依赖于TP53的方式上调，该结果已发表于《Cell Death & Dis》。同时发现，DR5缺失可导致结肠癌细胞对5-FU显著耐药。初步实验结果表明，这种耐药机制可能与Bid持续磷酸化使得Bid剪切受阻以及Bax活化受阻，最终导致caspase-3激活被抑制有关；DR5缺失可导致JNK激活受阻，JNK受阻与Bid剪接的减弱同样存在关联。本项目拟在此基础上，用CRISPR/Cas9系统构建不同敲除蛋白细胞系，用多种方法进一步从不同层面验证DR5与JNK活化以及Bid磷酸化状态之间的关联和机制，探明DR5缺失导致耐药的机制，并解析其临床意义。

Colorectal cancer is treated with various anti-cancer drug cocktails of which almost all include 5-Fluorouracil (5-FU). 5-FU exerts its effect by causing apoptosis in tumour cells. However, a problem in its clinical use is the development of chemo-resistance, which at the molecular level can be caused by apoptosis resistance. In previous study, we found selectively activate the DR5 by DR5-specific TRAIL with 5-FU has synergistic effects in inducing apotosis in colon cancer cells both in vitro and in vivo. Noteworthy, this effect was protein 53 (p53) independent and was mediated by DR5 up-regulation, demonstrating the applicability of this approach in p53-defective tumors. Meanwhile we found depletion of DR5 can cause resistance to 5-FU and the mechanisms are still unknown. we found that the characteristics of 5-FU-induced apoptosis in HCT116 cells are reminiscent of the death receptor-mediated apoptosis in so called type II cells where death receptor signaling to effector caspases is amplified by recruiting mitochondria into an amplification loop that involves Bid cleavage by caspase-8, Bax activation and the release of Smac/DIABLO. The intrinsic pathway is indispensible for 5-FU-induced apoptosis in HCT116 cells and depletion of DR5 can surprisingly cause the block of the intrinsic pathway. Further analysis revealed that the block of the intrinsic pathway was caused by inhibition of Bid cleavage and activation. Thus, the molecular point of resistance is the Bid protein. Furthermore, we also found that JNK is activated in the HCT wt cells, but not in HCT DR5-/- cells after 5-FU treatment. Based on those findings we proposed that JNK activation is DR5-dependent and might be involved in the process of regulating Bid phosphorylation. In order to test the hypothesis, we are going to test it at different levels and unveil its clinical implications.