DR5调节JNK通路及Bid磷酸化在结肠癌耐药中的分子作用机制及临床意义

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.

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。

本项目主要由以下四个部分组成：1. 研究了DR5在vernodalol增强TRAIL因子的细胞毒性中的作用，为揭示DR5在细胞耐药中的作用提供了一定的理论基础。本研究发现DR5的存在和JNK通路的激活存在密切，JNK通路的激活导致了转录因子CHOP的表达上调，而双荧光素报告实验则表明CHOP为DR5的转录因子，当使用RNAi沉默掉CHOP或者采用JNK通路抑制剂则显著抑制DR5表达上调。该结果发表于Mol Carcinog 56(10):2190-2199。2. 对ATR-1对膀胱癌细胞的杀伤作用及其机制进行了相关研究，我们发现ATR-1诱导的膀胱癌细胞凋亡和PI3K/Akt信号通路抑制密切相关，此外ATR-1还可以诱导细胞周期抑制于G2/M期，此研究结果发表于Journal of Experimental & Clinical Cancer Research (2016) 35:40。3. 为了更进一步研究DR5在肿瘤耐药以及死亡中的作用，我们发现Metformin和FTY720可以诱导DR5表达上调，此外Metformin和FTY720可以诱导ROS产生，ER 压力，并且ER压力和DR5表达上调之间存在有密切关联，此研究结果发表于Cell Physiol Biochem 2018;48:785-800。.4. 我们还就Camalexin对AML细胞的作用进行了研究，我们发现Camalexin可以明显减少AML细胞活性，增加ROS水平， SOD，CAT活性以及GSSG水平，而GSH水平则显著下降，并且导致ER压力，此外ROS水平的上升和ER压力存在有密切关联，而且ROS水平上升和DR5表达上调也存在关系，此研究结果发表于Oxidative Medicine and Cellular Longevity, 18

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