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

描述（由申请方提供）：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、糖尿病和胰腺炎是风险因素。这种癌症的预后非常差，因为它对现有疗法有抵抗力和广泛的转移。因此，迫切需要防治这种致命疾病的新战略，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。目前的项目利用我们的发现，β-肾上腺素受体调节人PDAC细胞系及其起源细胞，胰腺导管上皮细胞的生长。激动剂对这些G蛋白偶联受体的刺激通过腺苷酸环化酶=>cAMP=>PKA=>CREB诱导信号传导，并以PKA依赖性方式反式激活EGF途径。另外，异丙肾上腺素刺激细胞迁移，并具有很强的抗凋亡作用，如通过抑制饥饿诱导的切割的Casapse 3所证明的。GABA和巴氯芬对所有这些反应都有很强的抑制作用，并且还降低了未处理细胞的增殖和迁移。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的增加强烈刺激PDAC异种移植物的生长，诱导异种移植物细胞中的p-CREB和p-ERK 1/2，同时抑制GABA合成酶GAD 65和GABA。用GABA处理小鼠完全阻断异种移植物生长，同时使p-CREB、p-ERK 1/2、GAD和GABA的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的新型药物靶标。为了验证这一假设，我们提出了四个具体目标：具体目标1：在存在和不存在响应尼古丁和NNK的应激神经递质刺激的情况下，评价GABA和GABAB受体激动剂巴氯芬对裸鼠PDAC异种移植物发育早期和晚期的抗肿瘤作用。具体目标2：为了检验以下假设：在尼古丁和NNK处理的PDAC异种移植物中观察到的GAD 65和GABA抑制是由GAD 65的基因启动子超甲基化引起的，GABA逆转这些作用，并验证这些基因沉默和逆转机制在体外胰管上皮细胞中。具体目标3：为了检验以下假设：在体外和PDAC异种移植物中，在存在应激神经递质刺激的情况下，吉非替尼和吉西他滨对PDAC细胞的抗肿瘤作用降低，并且使用任何一种药物加GABA或巴氯芬的联合治疗可提高其有效性。具体目标4：评估β-受体阻滞剂普萘洛尔或考克斯-2抑制剂塞来昔布在体外和有和无应激神经递质刺激的PDAC异种移植物中的作用，并将其有效性与GABA和巴氯芬进行比较。该项目产生的数据可能导致以标记物指导的方式使用GABA能药物成功预防和治疗吸烟相关的PDAC，并更好地理解PDAC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非癌症疾病，因此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，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，由应激神经递质肾上腺素和去甲肾上腺素的增加引起的过度活跃的cAMP依赖性信号传导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。基于这一新概念，我们提出GABAB受体作为胰腺癌治疗和预防的新药物靶点。本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.