GABA-B-R-mediated prevention of pancreatic cancer

描述（由申请人提供）： 胰腺导管腺癌（PDAC）是癌症死亡的第四大原因，几乎100%的患者在诊断后6个月内死亡。因此，迫切需要预防PDAC的新策略。使用PDAC的两个危险因素（胰腺炎和吸烟），我们通过饮用水中的乙醇诱导动物胰腺炎，同时额外注射尼古丁衍生的致癌亚硝胺4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK），建立了PDAC的仓鼠模型。使用这种动物模型以及小鼠中来自人PDAC的异种移植物和使用人PDAC细胞系和PDAC的假定起源细胞胰腺导管上皮细胞的体外研究，我们发表的和新的初步数据显示，β-肾上腺素受体（b-AR）下游的腺苷酸环化酶依赖性细胞内信号传导刺激PDAC，同时另外触发表皮生长因子的释放，血管内皮生长因子和花生四烯酸。NNK通过与b-AR结合直接激活该信号通路，并通过刺激α 7烟碱乙酰胆碱受体（α 7 nAChR）介导的应激神经递质去甲肾上腺素和肾上腺素（它们是b-AR的激动剂）的释放间接激活该信号通路。该刺激网络的所有组分在PDAC中上调，而同时通过阻断腺苷酸环化酶的活化来抑制该途径的g-氨基丁酸（GABA）被抑制。用GABA处理体外PDAC细胞或体内PDAC异种移植物具有抑制作用。这些结果表明，GABA作为一个潜在的PDAC预防剂。为了验证这一假设，同时进一步了解PDAC通过刺激性b-AR信号和抑制性GABA信号的调节，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，我们将测试GABA或合成GABA类似物巴氯芬阻止PDAC发展的假设。具体目标二：使用PCR微阵列，蛋白质印迹和免疫组织化学，我们将调查的调制标记物的增殖，血管生成，转移，凋亡，细胞更新，和cAMP信号从仓鼠PDAC和正常胰腺组织中的GABA。具体目标3：使用人PDAC细胞系和体外胰腺导管上皮细胞，我们将确定GABA在b-AR和PGE 2介导的信号通路中的抑制作用，并探索GABA对TNF α和IL-1b的潜在降低作用。具体目标4：我们将检验以下假设：由于NNK诱导的α 4 nAChR的脱敏而抑制GABA的产生以及由于NNK诱导的α 7 nAChR的上调而刺激应激神经递质的产生有助于PDAC的刺激。所产生的数据将为使用GABA预防PDAC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，在诊断后一年内死亡率接近100%，因为它对现有疗法没有反应并且广泛转移。因此，迫切需要防治这一致命疾病的新战略。本项目中提供的数据沿着胰腺癌风险因素的已知生物学效应（吸烟、糖尿病、胰腺炎）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展和进展，同时胰腺产生γ-氨基丁酸（GABA），其通常通过激活抑制性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。本项目提出的体外和体内研究将为使用GABA能药物在胰腺GABA缺乏症患者中进行标记物引导的胰腺癌预防提供临床前基础。

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.