The GABA-B receptor is a novel drug target for pancreatic cancer

项目摘要。 胰腺导管腺癌（PDAC）是西方国家癌症死亡率的第四大原因 吸烟、糖尿病和胰腺炎是危险因素。这种癌症的预后非常差， 其对可用疗法的抗性和广泛的转移。对抗这种致命疾病的新策略 因此，迫切需要的，是国家癌症研究所确定的优先研究领域之一。 该项目的长期目标是开发新的和有效的治疗和预防策略 关于PDAC目前的项目利用了我们的发现，β-肾上腺素受体调节生长 人PDAC细胞系及其来源细胞，胰腺导管上皮细胞。刺激这些G- 激动剂通过腺苷酸环化酶=>cAMP=>PKA=>CREB诱导信号传导的蛋白偶联受体和 EGF的作用是通过依赖PKA的方式进行的。异丙肾上腺素额外刺激细胞 迁移，并有很强的抗凋亡作用，如抑制饥饿诱导的切割 casapse 3. GABA和巴氯芬对所有这些反应都有很强的抑制作用， 未处理细胞的增殖、迁移。为了支持这些体外研究结果，尼古丁诱导的 全身应激神经递质肾上腺素和去甲肾上腺素强烈刺激PDAC的生长 诱导异种移植细胞中p-CREB和p-ERK 1/2，同时抑制GABA合成 GAD 65和GABA。用GABA处理小鼠完全阻断异种移植物生长， 使p-CREB、p-ERK 1/2、GAD和GABA水平恢复到正常水平。这些数据表明GABAB 受体作为治疗和预防PDAC的新型药物靶点。为了验证这一假设，我们提出 四个具体目标： 具体目的1：评价GABA和GABAB受体激动剂巴氯芬的抗肿瘤作用 在存在和不存在PDAC的情况下，在裸鼠中PDAC异种移植物发育的早期和晚期阶段， 应激神经递质刺激对尼古丁和NNK的反应。 具体目的2：检验尼古丁和尼古丁受体中观察到的GAD 65和GABA抑制 NNK处理的PDAC异种移植物是由GAD 65的基因启动子超甲基化引起的，GABA逆转了GAD 65的基因启动子超甲基化。 这些作用，并验证这些机制的基因沉默和逆转在体外胰腺导管上皮细胞 细胞 具体目的3：检验吉非替尼和吉西他滨对PDAC的抗肿瘤作用 细胞在体外和PDAC异种移植物中存在应激神经递质刺激时减少， 用任何一种药剂加上GABA或巴氯芬的组合治疗提高了它们的有效性。 具体目的4：评估β受体阻滞剂普萘洛尔或考克斯-2抑制剂塞来昔布的体外作用 以及在有和没有应激神经递质刺激的PDAC异种移植物中，并比较它们的 与GABA和巴氯芬相似。 该项目产生的数据可能会导致成功预防和治疗吸烟- 以标记物引导的方式将PDAC与GABA能药物相关联，并更好地了解 在PDAC的调节中刺激和抑制神经递质的复杂作用机制。 由于GABA和巴氯芬已经被批准用于治疗人类的非癌性疾病， 这项研究的结果可以迅速转化为临床试验。

Project Summary. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in Western countries and smoking, diabetes, and pancreatitis are risk factors. The prognosis of this cancer is extremely poor due to its resistance to available therapies and extensive metastasis. New strategies to combat this deadly disease are thus urgently needed and are among priority areas of research identified by the National Cancer Institute. The long-term goal of this project is to develop novel and effective strategies for the treatment and prevention of PDAC. The current project takes advantage of our discovery that beta-adrenoreceptors regulate the growth of human PDAC cell lines and their cells of origin, pancreatic duct epithelial cells. Stimulation of these G- protein coupled receptors by agonists induced signaling via adenylyl cyclase=>cAMP=>PKA=>CREB and transactivated the EGF pathway in a PKA-dependent manner. Isoroterenol additionally stimulated cell migration and had strong anti-apoptotic effects as evidenced by suppression of starvation-induced cleaved casapse 3. GABA and baclophen had strong inhibiting effects on all these responses and additionally reduced proliferation, migration of untreated cells. In support of these in vitro findings, nicotine-induced increase in systemic stress neurotransmitters adrenaline and noradrenaline strongly stimulated the growth of PDAC xenografts, induced p-CREB and p-ERK1/2 in xenograft cells while suppressing the GABA synthesizing enzyme GAD65 and GABA. Treatment of the mice with GABA completely blocked xenograft growth while returning levels of p-CREB, p-ERK1/2, GAD and GABA to normal levels. These data suggest the GABAB receptor as a novel drug target for the treatment and prevention of PDAC. To test this hypothesis we propose four specific aims: Specific aim 1: To evaluate the anti-tumorigenic effects of GABA and the GABAB receptor agonist baclophen on early and advanced stages of PDAC xenograft development in nude mice in the presence and absence of stress neurotransmitter stimulation in response to nicotine and NNK. Specific aim 2: To test the hypothesis that the observed suppression of GAD65 and GABA in nicotine and NNK-treated PDAC xenografts is caused by gene promoter hypermethylation of GAD65, that GABA reverses these effects and to verify these mechanism of gene silencing and reversal in vitro in pancreatic duct epithelial cells. Specific aim 3: To test the hypothesis that the antitumorigenic effects of gefitinib and gemcitabine on PDAC cells are reduced in the presence of stress neurotransmitter stimulation in vitro and in PDAC xenografts and that combination treatments with either agent plus GABA or baclophen improves their effectiveness. Specific aim 4: To assess the effects of the beta-blocker propranolol or the COX-2 inhibitor celecoxib in vitro and in PDAC xenografts with and without stimulation by stress neurotransmitters, and to compare their effectiveness to that of GABA and baclophen. Data to be generated by this project may lead to the successful prevention and treatment of smoking- associated PDAC in a marker-guided fashion with GABA-ergic agents and generate a better understanding of the complex mechanisms of action of stimulating and inhibiting neurotransmitters in the regulation of PDAC. Since GABA and baclophen are already approved for the treatment of non-cancerous conditions in humans, results of this research can be rapidly translated into clinical trials.