GABA-B-R-mediated prevention of pancreatic cancer

描述(由申请人提供)： 胰腺导管腺癌(PDAC)是导致癌症死亡的第四大原因，近100%的患者在确诊后6个月内死亡。因此，迫切需要预防PDAC的新战略。利用诱发PDAC的两种危险因素(胰腺炎和吸烟)，我们建立了PDAC的仓鼠模型，方法是用饮用水中的乙醇诱发动物胰腺炎，同时再注射尼古丁衍生的致癌亚硝胺4-(甲基亚硝胺)-1-(3-吡啶)-1-丁酮(NNK)。利用该动物模型和人PDAC在小鼠体内的异种移植，以及对人PDAC细胞系和PDAC可能起源的细胞-胰管上皮细胞的体外研究，我们发表的新的初步数据显示，β-肾上腺素受体(b-ARs)下游的腺酰环化酶依赖的细胞内信号在刺激PDAC的同时还触发了表皮生长因子、血管内皮生长因子和花生四烯酸的释放。NNK通过直接与b-AR结合来激活这一信号通路，并通过刺激a7nAChR介导的应激神经递质去甲肾上腺素和肾上腺素的释放间接激活这一信号通路，这两种递质是b-AR的激动剂。在PDAC中，这个刺激网络的所有成分都被上调，而g-氨基丁酸(GABA)则被抑制，后者通过阻断腺苷环化酶的激活来抑制这一途径。GABA对体外培养的PDAC细胞或体内移植的PDAC细胞均有抑制作用。这些发现表明GABA是一种潜在的PDAC预防药物。为了验证这一假说，同时加深我们对刺激的b-AR信号和抑制的GABA信号对PDAC的调控的理解，我们提出了四个具体的目标。具体目标1：使用我们的仓鼠模型，我们将检验GABA或合成的GABA类似物巴洛芬阻止PDAC发生的假设。具体目的2：利用基因芯片、Western blotting和免疫组织化学方法，研究GABA对仓鼠胰腺组织和正常胰腺组织中增殖、血管生成、转移、细胞凋亡、细胞更新和cAMP信号转导等标志物的调控作用。具体目的3：利用体外培养的人PDAC细胞株和胰管上皮细胞，研究GABA对b-AR和PGE2介导的信号通路的抑制作用，探讨GABA对肿瘤坏死因子α(TNFa)和白介素1b(IL-1b)的抑制作用。具体目标4：我们将检验NNK诱导的a4nAChR脱敏而抑制GABA的产生和NNK诱导的a7nAChR上调而刺激应激性神经递质产生的假说有助于刺激PDAC。产生的数据将为GABA在预防PDAC中的使用提供临床前基础。 公共卫生相关性： 胰腺癌是癌症死亡的第四大原因，确诊后一年内死亡率接近100%，因为它对现有的治疗方法没有反应，而且会广泛转移。因此，迫切需要新的战略来抗击这种致命的疾病。该项目提供的数据以及胰腺癌危险因素(吸烟、糖尿病、胰腺炎)的已知生物学效应表明，过度活跃的β-肾上腺素受体和可能的其他G蛋白偶联受体刺激胰腺癌的发生和发展，同时胰腺产生的伽马氨基丁酸(GABA)减少，通常通过激活抑制的GABAB受体来控制这些受体的活性。基于这一新的概念，我们建议恢复胰腺GABA的缺陷，以预防胰腺癌。该项目提出的体外和体内研究将为使用GABA能药物用于标记物导向预防胰腺GABA缺乏个体的胰腺癌提供临床前基础。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality with near 100% of the victims succumbing within 6 month of diagnosis. New strategies for PDAC prevention are therefore urgently needed. Using two risk factors for PDAC (pancreatitis and smoking), we have established a hamster model of PDAC by inducing pancreatitis in the animals via ethanol in the drinking water while additionally injecting them with the nicotine-derived carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). Using this animal model as well as xenografts from human PDAC in mice and in vitro studies with human PDAC cell lines and the putative cell of origin of PDAC, pancreatic duct epithelial cells, our published and new preliminary data show that adenylyl cyclase-dependent intracellular signaling downstream of beta-adrenoreceptors (b-ARs) stimulates PDAC while additionally triggering the release of epidermal growth factor, vascular endothelial growth factor and arachidonic acid. NNK activates this signaling pathway directly by binding to b-ARs and indirectly by stimulating the a7nicotinic acetylcholine receptor (a7nAChR)-mediated release of the stress neurotransmitters noradrenaline and adrenaline, which are agonists for b-ARs. All components of this stimulatory network are upregulated in PDAC while at the same time g-aminobutyric acid (GABA) which inhibits this pathway by blocking the activation of adenylyl cyclase is suppressed. Treatment of PDAC cells in vitro or PDAC xenografts in vivo with GABA had inhibiting effects. These findings suggest GABA as a potential PDAC preventive agent. To test this hypothesis and at the same time further our understanding on the regulation of PDAC by stimulatory b-AR signaling and inhibitory GABA signaling we propose four specific aims. Specific Aim 1: Using our hamster model, we will test the hypothesis that GABA or the synthetic GABA analogue baclophen prevent the development of PDAC. Specific Aim 2: Using PCR microarrays, Western blotting and immunohistochemistry, we will investigate the modulation of markers for proliferation, angiogenesis, metastasis, apoptosis, cell renewal, and cAMP signaling by GABA in tissues from hamster PDAC and normal pancreatic tissue. Specific Aim 3: Using human PDAC cell lines and pancreatic duct epithelial cells in vitro, we will determine the inhibitory actions of GABA in b-AR and PGE2-mediated signaling pathways and explore a potential reduction in TNFa and IL-1b by GABA. Specific Aim 4: We will test the hypothesis that inhibited GABA production due to NNK-induced desensitization of the a4nAChR and stimulation of stress neurotransmitter production due to NNK-induced upregulation of the a7nAChR contribute to the stimulation of PDAC. Data generated will provide a preclinical basis for the use of GABA in the prevention of PDAC. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is the fourth leading cause of cancer death with a mortality near 100% within one year of diagnosis because it does not respond to existing therapies and metastasizes extensively. New strategies to combat this deadly disease are thus urgently needed. Data presented in this project along with known biological effects of risk factors for pancreatic cancer (smoking, diabetes, pancreatitis) suggest that hyperactive beta-adrenoreceptors and possibly other G-protein coupled receptors stimulate pancreatic cancer development and progression while at the same time the pancreatic production of gamma-aminobutyric acid (GABA), which normally controls the activity of these receptors via activation of the inhibitory GABAB receptor, is reduced. Based on this novel concept, we propose to restore deficient pancreatic GABA for the prevention of pancreatic cancer. In vitro and in vivo studies proposed under this project will provide a preclinical basis for the use of GABA-ergic agents for the marker-guided prevention of pancreatic cancer in individuals with pancreatic GABA deficiency.