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

描述(申请人提供)：胰腺导管腺癌(PDAC)是西方国家癌症死亡的第四大原因，吸烟、糖尿病和胰腺炎是危险因素。由于对现有治疗方法的抵抗和广泛的转移，这种癌症的预后非常差。因此，迫切需要防治这一致命疾病的新战略，这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治疗和预防PDAC的新的有效策略。目前的项目利用了我们的发现，即β-肾上腺素受体调节人PDAC细胞系及其来源细胞-胰管上皮细胞的生长。激动剂刺激这些G蛋白偶联受体，通过腺苷环化酶cAMP=&gt；PKA=&gt；CREB诱导信号转导，并以PKA依赖的方式反式激活EGF途径。异松质醇还能刺激细胞迁移，并有很强的抗凋亡作用，表现为抑制饥饿诱导的裂解Casapse 3。GABA和巴洛芬对所有这些反应都有很强的抑制作用，还能减少未经处理的细胞的增殖和迁移。为支持这些体外实验结果，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的增加强烈刺激了PDAC移植瘤的生长，诱导了异种移植细胞中的p-CREB和p-ERK1/2，同时抑制了GABA合成酶GAD65和GABA。用GABA治疗小鼠完全阻止了异种移植瘤的生长，同时使p-CREB、p-ERK1/2、GAD和GABA水平恢复到正常水平。这些数据表明GABAB受体是治疗和预防PDAC的一个新的药物靶点。为了验证这一假设，我们提出了四个特定的目标：特定的目标1：在尼古丁和NNK的应激性神经递质刺激和不存在应激性神经递质刺激的情况下，评估GABA和GABAB受体激动剂巴洛芬对PDAC裸鼠移植瘤早期和晚期的抗肿瘤作用。特定目的2：验证尼古丁和NNK处理的PDAC移植瘤中GAD65和GABA的抑制是由GAD65基因启动子超甲基化引起的假设，GABA逆转这些作用，并在体外验证这些基因沉默和逆转的机制。具体目的3：验证如下假设，即在体外和PDAC异种移植中，在应激性神经递质刺激存在的情况下，吉非替尼和吉西他滨对PDAC细胞的抗肿瘤作用降低，而两种药物中的任何一种与GABA或巴洛芬联合治疗均可提高其疗效。具体目的4：评价β受体阻滞剂普萘洛尔或环氧合酶-2抑制剂塞来昔布在体外和在有或无应激神经递质刺激的PDAC异种移植中的作用，并与GABA和巴洛芬的效果进行比较。该项目将产生的数据可能导致以标记物引导的方式使用GABA能药物成功地预防和治疗吸烟相关性PDAC，并使人们更好地理解刺激和抑制神经递质在PDAC调节中的复杂作用机制。由于GABA和巴洛芬已经被批准用于治疗人类的非癌症疾病，这项研究的结果可以迅速转化为临床试验。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，并广泛转移。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已知的胰腺癌危险因素(吸烟、糖尿病、胰腺炎)的生物学效应表明，应激神经递质肾上腺素和去甲肾上腺素增加导致的cAMP依赖过度激活的信号通路刺激胰腺癌的发生和进展，同时胰腺产生的伽马氨基丁酸(GABA)减少，通常通过激活抑制性GABAB受体来控制这些受体的活性。基于这一新概念，我们提出了GABAB受体作为治疗和预防胰腺癌的新的药物靶点。该项目提出的体外和体内研究将为使用GABA能药物有效治疗和预防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 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. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is the fourth leading cause of cancer death with 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 cAMP-dependent signaling caused by an increase in the stress neurotransmitters adrenaline and noradrenaline stimulates 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 the GABAB receptor as a novel drug target for the therapy and 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 effective therapy and prevention of pancreatic cancer.