The Role of t-Darpp in Esophageal Adenocarcinoma

描述(申请人提供)：上胃肠腺癌(UGCS；食道腺癌和胃腺癌)的发病率一直在稳步上升。此外，在过去三十年中，食道下段和胃食道交界处腺癌的发病率急剧上升，这是西方世界所有癌症中发病率上升最快的。总体相对5年存活率目前不到20%。改善我们目前有限的对UGCS的诊断、预防和治疗方法是目前一个紧迫的问题。这一建议是基于我们最初的发现，通过使用UGCS的多个分子分析，导致克隆和鉴定了t-DARPP这一新的癌症基因。我们发现t-DARPP在大约三分之二的UGC中高表达，对其生物学功能的分析表明，t-DARPP是一种有效的促生存蛋白。使用几种药物(喜树碱、丁酸盐、神经酰胺)，我们发现t-DARPP通过激活AKT生存通路和上调bcl2来保护癌细胞免受药物诱导的细胞死亡。我们假设，t-DARPP为癌细胞提供了关键的、强有力的促进生存和抗凋亡的优势，从而导致了对药物诱导的细胞死亡的抵抗。我们计划研究t-DARPP的分子和生物学作用，并确定其敲除在UGCS中的潜在治疗价值。在本提案的目标1中，我们将研究t-DARPP磷酸化位点调节AKT存活途径的分子机制(S)。基于我们的初步数据，我们将研究t-DARPP调节PTEN抑癌基因在癌细胞中的活性、定位和稳定性的机制。在目标2中，我们计划通过测试t-DARPP消除药物诱导的细胞死亡的能力来研究其对耐药的影响。我们将研究t-DARPP在调节复杂的内源性细胞凋亡中的作用。最近的一些报告表明癌基因成瘾的概念在癌细胞中，其中癌细胞的生存变得依赖于一种关键的生存蛋白的表达。我们将在体内测试t-DARPP基因敲除的治疗潜力，以及它是否单独或与现有化疗药物联合使用可以提高治疗反应。在目标3中，我们将采用强大的蛋白质组学方法对与t-DARPP相互作用的蛋白质和那些下游效应蛋白进行系统表征，以确定t-DARPP可能在未知的癌细胞中介导的新的信号通路和生物学功能。在我们的所有实验中，我们将测试其磷酸化突变体，以表征每个磷酸化位点在癌细胞中的分子和功能作用(S)。我们预计，建议实验的完成将为t-DARPP作为一种新的蛋白质在上胃肠腺癌中的作用提供重要的洞察力。公共卫生相关性：这项建议连接了体外和体内实验，以表征t-DARPP在上消化道癌变中的作用(S)。我们计划研究t-DARPP在调节内源性细胞凋亡中的作用，并评估其作为新的治疗靶点的潜力。最先进的蛋白质组学方法将被用来鉴定t-DARPP相互作用的蛋白质及其分子信号靶标。

DESCRIPTION (provided by applicant): The incidence of upper gastrointestinal adenocarcinomas (UGCs; adenocarcinomas of the esophagus and stomach) has been rising steadily. Moreover, a sharp increase in the incidence of lower esophageal and gastroesophageal junction adenocarcinomas has been observed over the past three decades bearing the distinction of the fastest rising incidence of all cancers in the Western world. Overall relative 5-year survival rates are currently less than 20%. Improvement in our presently limited diagnostic, preventive, and therapeutic approach to UGCs is currently a pressing issue. This proposal is based on our original findings through the use of multiple molecular analyses in UGCs that led to the cloning and identification of t-DARPP as a novel cancer gene. We found over-expression of t-DARPP in approximately two-thirds of UGCs and analysis of its biological functions indicated that t-DARPP is a potent pro-survival protein. Using several drugs (camptothecin, butyrates, ceramides) we found that t-DARPP protects cancer cells against drug-induced cell death by a mechanism that includes activation of AKT survival pathway and up-regulation of BCL-2. We hypothesize that t-DARPP provides critical potent pro-survival and anti-apoptotic advantages to cancer cells leading to resistance to drug- induced cell death. We plan to investigate the molecular and biological roles of t-DARPP and determine the potential therapeutic value of its knockdown in UGCs. In Aim 1 of this proposal, we will investigate the molecular mechanism(s) by which t-DARPP phosphorylation sites regulate the AKT survival pathway. Based on our preliminary data, we will investigate the mechanisms by which t-DARPP regulates the PTEN tumor suppressor activity, localization, and stability in cancer cells. In Aim 2, we plan to investigate the effect of t- DARPP on drug resistance by testing its ability to abrogate drug-induced cell death. We will examine the role of t-DARPP in regulating the complex intrinsic apoptosis. Several recent reports suggest the oncogene addiction concept in cancer cells, where cancer cell survival becomes dependent on the expression of a critical survival protein. We will test the therapeutic potential of t-DARPP knockdown in vivo and whether its knockdown alone or in combination with the existing chemotherapeutics can boost the therapeutic response. In Aim 3, we will adopt powerful proteomic approaches for a systematic characterization of proteins that interact with t-DARPP and those that are downstream effectors in order to identify novel signaling pathways and biological functions that t-DARPP could mediate in cancer cells that remain unknown. In all our experiments, we will test its phosphorylation mutants in order to characterize the molecular and functional role(s) of each of these phosphorylation sites in cancer cells. We anticipate that completion of the suggested experiments will provide a significant insight into the role of t-DARPP, as a novel protein, in upper gastrointestinal adenocarcinomas. PUBLIC HEALTH RELEVANCE: This proposal connects in vitro and in vivo experiments in order to characterize the role(s) of t-DARPP in upper gastrointestinal carcinogenesis. We plan to investigate the role of t-DARPP in regulating the intrinsic apoptosis and evaluate its potential as a novel therapeutic target. State-of-the-art proteomic approaches will be employed to identify t-DARPP interacting proteins as well as its molecular signaling targets.