Inhibition of pancreatic carcinogenesis via targeting c-Raf and sEH

项目描述（由申请人提供）：本项目的意义在于开发一种同时靶向c-Raf和sEH的高效预防高致死性胰腺癌的药物。早期发现和预防对降低胰腺癌的死亡率具有重要意义。Kras突变是人类胰腺癌发生中最常见和最早的分子事件；并在小鼠中使用突变的Kras启动的肿瘤发生，已经证明c-Raf是Kras启动的肿瘤发生所必需的，而敲除c-Raf可阻断Kras启动的癌症发展。慢性胰腺炎是公认的胰腺癌的危险因素。在Pdx1-Cre/LSL-KrasG12D小鼠（称为PanKras）中突变型kras引发的胰腺肿瘤发生中，小蛋白诱导的胰腺炎促进恶性进展。除了胰腺炎，Kras突变体与p53突变体（第二常见的基因改变）合作，主要通过协同调节ERK-MAPK和NF-kB，导致小鼠胰腺癌的发展。抗炎eet被促炎可溶性环氧化物水解酶（sEH）迅速灭活。我们的初步数据显示，1)96%（27/28）和90%（27/30）的人胰腺腺癌样本分别表现出明显的过表达sEH和p-c-Raf， 2)我们的新型双c-Raf/sEH抑制剂t-CUPM对PanKras小鼠的胰腺炎和mPanIN的形成有很强的抑制作用。我们的假设是c-Raf和sEH在突变Kras引发的胰腺癌发生中发挥关键作用，并在胰腺炎或突变p53的作用下增强；同时靶向这两种酶是一种新的、相关的胰腺癌预防策略。为了验证这一假设，我们提出了三个具体目标：1)确定c-Raf/sEH双抑制剂t- cupm、sEH抑制剂t- AUCB和Raf抑制剂Raf265/sorafenib对突变型kras引发的PanKras小鼠和胰腺炎增强的癌变的影响；2)确定在Pdx1-KrasG12D-p53R172小鼠中，使用抑制剂或基因敲除阻断突变型kras活化的c-Raf是否会抑制突变型Kras-p53相互作用和恶性进展；3)通过敲除PanKras小鼠的sEH基因，确定sEH在胰腺炎和癌变中的作用。本研究的成功不仅可以证明c-Raf和sEH在胰腺癌发生中的作用，还可以确定t-CUPM作为抑制胰腺癌发展的新药物的意义。

DESCRIPTION (provided by applicant): The significance of this project is to develop an efficient agent for the prevention of highly lethal pancreatic cancer through targeting both c-Raf and sEH. Early detection and prevention is of great importance to reduce the mortality of pancreatic cancer. Mutant Kras is the most common and earliest molecular event involved in human pancreatic carcinogenesis; and using mutant Kras-initiated tumorigenesis in mice, it has been demonstrated that c-Raf is required for Kras-initiated tumorigenesis and knockout c-Raf blocks Kras- initiated cancer development. Chronic pancreatitis is a well-recognized risk factor of pancreatic cancer. In mutant Kras-initiated pancreatic tumorigenesis in Pdx1-Cre/LSL-KrasG12D mice (called PanKras), caerulein- induced pancreatitis enhances malignant progression. In addition to pancreatitis, mutant Kras cooperates with mutant p53 (the second most common genetic alteration) leading to pancreatic cancer development in mice, mainly via synergistically regulating ERK-MAPK and NF-kB. Anti-inflammatory EETs are quickly inactivated by pro-inflammatory soluble epoxide hydrolase (sEH). Our preliminary data showed that 1) 96% (27/28) and 90% (27/30) of human pancreatic adenocarcinoma samples displayed a distinct over-expression of sEH and p-c-Raf, respectively, and 2) our novel dual c-Raf/sEH inhibitor t-CUPM exhibited strong inhibitory effect on pancreatitis and mPanIN formation in PanKras mice. Our hypothesis is that c-Raf and sEH play key roles in pancreatic carcinogenesis initiated by mutant Kras and enhanced by pancreatitis or mutant p53; and targeting these two enzymes together is a novel and relevant preventive strategy for pancreatic cancer. Three specific aims are proposed to test this hypothesis: 1) to determine the effects of c-Raf/sEH dual inhibitor t-CUPM, sEH inhibitor t- AUCB and Raf inhibitor Raf265/sorafenib on mutant Kras-initiated and pancreatitis-enhanced carcinogenesis in PanKras mice; 2) to determine whether blocking mutant Kras-activated c-Raf using inhibitors or gene knockout suppresses mutant Kras-p53 interaction and malignant progression in Pdx1-KrasG12D-p53R172 mice; and 3) to determine the role of sEH in pancreatitis and carcinogenesis using the approach of sEH gene knockout in PanKras mice. Successfulness of the proposed study will not only lead us to demonstrate the role of c-Raf and sEH in pancreatic carcinogenesis, but also to establish a significance of t-CUPM as a novel agent for inhibiting the pancreatic cancer development.