K-RAS活性调节在DJ-1促进胰腺癌侵袭中的作用及机制研究

A major hallmark of pancreatic ductal adenocarcinoma (PDAC) is extensive local tumor invasion and early systemic dissemination. Our previous study showed that DJ-1 was overexpressed in pancreatic cancer cells and was correlated with higher tumor T stage and shorter overall survival. In vivo and vitro results showed that DJ-1 promoted pancreatic cancer invasion through activation of ERK1/2 pathway and uPA upregulation. Further study showed that RAS activity which is the upstream of ERK1/2 was also regulated by DJ-1. Because K-RAS is commonly mutated in PDAC and invovled in PDAC progression, it is proposed that DJ-1 activates K-RAS, leading to activation of ERK1/2- uPA- invasion cascade.Therefore,in present study, we firstly aimed to confirm whether DJ-1 activates ERK1/2-uPA pathway via K-RAS. Secondly, the potential mechanisms by which DJ-1 regulates K-RAS activity will also be explored in the following fields: physical interaction between K-RAS and DJ-1, K-RAS lipid modification regulation, K-RAS transmembrane regulation, K-RAS activator and inhibitor protein (GAPs, GEFs) expression.The study will provide rationale for targeting DJ-1 treatment of pancreatic cancer, and help to find an effective strategy of targeting K-RAS.

高转移性和强侵袭性是胰腺癌重要特征。我们前期研究发现DJ-1在胰腺癌中高表达，且与肿瘤T分期及预后相关；并证实DJ-1通过激活ERK通路,上调uPA表达，促进胰腺癌侵袭转移；而ERK上游的RAS活性也受DJ-1调节。鉴于K-RAS在胰腺癌中高突变率，与胰腺癌密切相关，我们推测DJ-1通过增加K-RAS活性激活ERK-uPA-侵袭级联反应。故为明确DJ-1促胰腺癌侵袭作用是否依赖K-RAS，我们拟在敲低DJ-1表达细胞中转染组成型激活K-RAS，在高表达DJ-1细胞中转染siRNA抑制K-RAS活性，观察ERK通路及侵袭变化；其次为探讨DJ-1调节K-RAS活性机制，我们将检测DJ-1与K-RAS物理结合方式与位点、检测改变DJ-1表达后K-RAS在细胞内膜定位、脂质修饰水平及K-RAS激活和抑制蛋白表达量等变化。该研究为靶向DJ-1治疗胰腺癌提供理论依据，为寻找靶向K-RAS提供有效策略。

高转移性和强侵袭性是胰腺癌重要特征。我们前期研究发现DJ-1在胰腺癌中高表达，且与肿瘤T分期及预后相关；并证实DJ-1通过激活ERK通路,上调uPA表达，促进胰腺癌侵袭转移。本项目就DJ-1引起ERK通路上游即RAS的激活做了深入的研究，并据此联系临床实际探索这一机制对胰腺癌厄罗替尼的增敏效应。.结果主要发现：1） 干扰DJ-1表达后RAS各个类型亚型（KRAS、NRAS、HRAS）其活性均下降。2）干扰DJ-1后各个RAS的mRNA表达水平略有减低。3）干扰DJ-1抑制干扰DJ-1后细胞膜Kras/胞浆水平明显减低（减低44.1%，P＜0.001,N=3），HRAS和NRAS变化不明显。提示DJ-1可能参与K-RAS的膜转位。免疫荧光也证实干扰DJ-1 对KRAS在细胞内分布变化。4）研究提示干扰DJ-1后K-Ras的降解及合成速率影响未见明显变化。5)干扰DJ-1后RASAL1表达明显升高，RASAL1能催化RAS-GTP水解失活。6）干扰DJ-1减低K-RAS表达，抑制了RAS和K-RAS活性,可增强厄洛替尼对胰腺癌细胞的抑制增殖、促进凋亡的作用。本项目研究揭示了DJ-1调节KRAS活性具体机制可能通过增加KRAS的膜转位、mRNA表达，上调GAPs：RASAL1。.KRAS是目前胰腺癌的抗EGFR治疗耐药性重要下游分子，KRAS的状态影响了抗EGFR治疗的敏感性，我们的研究也显示抑制DJ-1可以增强抗EGFR抑制剂的疗效。因此，以上研究成果有望为胰腺癌的分子靶向治疗提供理论依据，尤其是为开发抗KRAS治疗药物，和联合抗EGFR治疗策略提供理论基础。