DUSP12介导的内质网应激在诱导TRAIL耐药胰腺癌细胞凋亡的机制研究

Pancreatic cancer is related with poor prognosis. Breakthrough drug resistance for pancreatic cancer is becoming a hot topic in medicine filed. Our previous research found that down-regulation of DUSP12 induces TRAIL resistance pancreatic cancer cells apoptosis. However, the detailed mechanism is still unclear. Further research indicated that DUSP12 is a dual specificity phosphatase, which is overexperssed in pancreatic cancer cells. The functional binding site of DUSP12 is similar to the phosphorylation site of PERK. We have proved that down-regulation of DUSP12 could inhibit its dephosphorylation, which initiates a key step of Endoplasmic Reticulum Stress: activation of PERK-eIF2. And the activaiton of PERK-eIF2 regulates death receptor signals through transcriptional factor: ATF-4, which further sensitizes TRAIL. Therefor, we provide the hypothesis as follows, DUSP12 bind with PERK and form a stable functional complex in TRAIL resistance pancreatic cancer cells. Down-regulaiton of DUSP12 by Triptolide activate death receptor signals through phosphorylation of ER stress key factor: PERK-eIF2. This project elaborates the mechanism of regulation of death receptor signals by Triptolide through oncogene DUSP12---PERK-eIF2 signal pathway. Since TRAIL is already in use against several cancers, understanding the mechanism by which Triptolide sensitizes pancreatic cancer cells to TRAIL may result in a novel therapeutic strategy against pancreatic cancer.

如何突破胰腺癌耐药屏障是目前药物治疗领域的热点。我们前期发现，Triptolide通过下调DUSP12致敏TRAIL，诱导胰腺癌细胞凋亡，而机制不明。文献报道，DUSP12是双向特异性去磷酸化酶，在胰腺癌中高表达，其功能结合位点与PERK的磷酸化结构域有高度同源性。预实验证实，下调DUSP12水平抑制其去磷酸化作用，启动内质网应激的关键步骤：PERK-eIF2激活，而该信号通路可通过转录因子ATF-4对死亡受体信号分子调控，从而致敏TRAIL。据此，我们提出在TRAIL耐药胰腺癌中，DUSP12与PERK形成功能复合体。当Triptolide下调DUSP12后，解除其去磷酸化作用，诱导PERK自磷酸化，通过eIF2激活下游转录因子ATF-4，调控死亡受体相关信号分子，致敏TRAIL的假说。本研究有望阐明Triptolide突破胰腺癌细胞TRAIL耐受屏障的新机制，为胰腺癌的治疗提供新思路。

背景：胰腺癌是一种恶性程度极高的肿瘤，我们前期研究发现Pumilio RNA-binding family member 1 (PUM1)基因在胰腺癌组织中异常表达。在此，我们旨在进一步研究其在胰腺癌中的作用及其发挥作用的方式。方法：采用免疫组法明确蛋白的表达差异，采用CCK-8检测细胞增殖；采用免疫印迹法检测蛋白表达；采用Transwell检测细胞迁移、侵袭水平。结果：PUM1蛋白的高表达预示着患者预后不良。抑制PUM1的表达可以抑制胰腺癌细胞的增殖、迁移、侵袭、上皮间质转化（epithelial–mesenchymal transition ，EMT），促进其凋亡。体内裸鼠皮下成瘤实验证明敲降PUM1可以抑制皮下肿瘤的生长，肺癌转移模型证明敲降PUM1可以减少PUM1肺组织中ki67阳性细胞的数量。PUM1的表达水平与PERK成正相关，在PUM1敲降的胰腺癌细胞中使用PERK的抑制剂可以部分逆转PUM1对胰腺癌细胞增殖、迁移、侵袭、EMT、凋亡的影响。微血管密度(microvessel density, MVD)值与PUM1蛋白表达呈正相关。人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）与过表达PUM1的胰腺癌细胞共培养时，其增殖、迁移、侵袭和成管能力增强。PUM1过表达增加胰腺癌细胞胞外和胞内VEGFA蛋白水平。此外，当HUVECs与过表达PUM1的胰腺癌细胞共培养时，HUVECs中血管生成相关信号被激活。然而，敲降PUM1的胰腺癌细胞却有相反的效果。此外，过表达PUM1的皮下移植瘤CD31表达水平较高，而敲降PUM1的皮下移植瘤CD31表达水平较低。结论：PUM1的下调可以通过PERK/eIF2/ATF4信号通路，抑制胰腺癌细胞的生长、侵袭、迁移、EMT，促进细胞的凋亡。同时，PUM1在胰腺癌中可能起到促进胰腺癌血管生成的作用。PUM1可能是一种新的胰腺癌血管生成调节因子。