EPIDERMAL GROWTH FACTOR RECEPTORS AND PANCREATIC CANCER

描述：(申请人摘要)胰腺导管腺癌(PDAC)是 一种致命的疾病。决定它的分子机制 生物攻击性尚不清楚。我们确定PDAC 肿瘤细胞高水平表达表皮生长因子受体 (EGFR)及其相关受体(ErbB-2、-3、-4)。我们现在假设 EGFR家族的过度激活在根本上促进了 PDAD的病理生物学。为了验证这一假设，我们将阻断受体 在培养的胰腺癌细胞中通过这个家族的每个成员的信号 线，使用高度具体的显性-否定性方法，结合 我们最近建立的腺病毒基因传递系统。信令将是下一个 通过此家族的多个成员进行拦截，以确定 信号通路被单一受体阻滞剂与联合受体阻滞剂相比减弱， 哪些途径调节有丝分裂，哪些途径对失巢凋亡具有抵抗力。在……里面 体内，我们将评估受体阻断对肿瘤生长和 以确定EGFR家族的过度激活是否为转移 信号转导与PDAC的这些生物学特性有关。转到更多 明确定义调节EGFR家族行动的信号组件，我们 将使用显性负向结构和化学抑制剂来抑制 这些通路的特定下游组件，并构建编码 以构成方式活跃的蛋白质。定义新的信令 受EGFR调控的途径，我们将使用中国仓鼠卵巢细胞 它们有相对正常的基因背景，没有内源 EGFR，但已经稳定地转染了编码野生型或 变异型人类EGFR。因此，我们将深入了解细胞的生物学作用。 这些高度同源的受体涉及有丝分裂、失巢凋亡和 侵犯性。评估PDAC中受体异二聚化的潜力 在体内，我们将使用激光捕获显微切割和定量聚合酶 用链式反应(PCR)检测同一癌细胞中的表达水平 所有四名EGFR家族成员的名字。如果我们把基因扩增排除为 体内过表达的机制，我们将确认我们的培养细胞 细胞系过度表达这些受体是增强转录的结果 核子研究。然后我们将描述它们转录控制的特征 以开发第二代病毒载体，这些病毒载体 优先靶向胰腺癌细胞。

DESCRIPTION: (Applicant's abstract) Pancreatic ductal adenocarcinoma (PDAC) is a devastatingly lethal disease. The molecular mechanisms that dictate its biological aggressiveness are yet to be elucidated. We determined that PDAC tumor cells express high levels of the epidermal growth factor (EGF) receptor (EGFR) and related receptors (ErbB-2, -3, -4). We now hypothesize that excessive activation of the EGFR family contributes in a fundamental manner to the pathobiology of PDAD. To test this hypothesis, we will block receptor signaling through each member of this family in cultured pancreatic cancer cell lines, using a highly specific dominant-negative approach in conjunction with our recently established adenoviral gene delivery system. Signaling will next be blocked through multiple members of this family in order to determine which signaling pathways are attenuated by single versus combined receptor blockades, which pathways modulate mitogenesis and which confer resistance to anoikis. In vivo, we will assess effects of receptor blockade on tumor growth and metastasis in order to determine whether excessive activation of EGFR family signaling contributes to these biological characteristics of PDAC. To more clearly define the signaling components that mediate EGFR family actions, we will use dominant-negative constructs and chemical inhibitors to suppress specific downstream components of these pathways, and constructs encoding proteins that are active in a constitutive manner. To define novel signaling pathways that are modulated by EGFR, we will use Chinese hamster ovary cells that have a relatively normal gene background and that are devoid of endogenous EGFR, but that have been stably transfected with a cDNA encoding a wild type or variant human EGFR. We will thus gain insight into the biological roles of these highly homologous receptors with respect to mitogenesis, anoikis and invasiveness. To assess the potential for receptor heterodimerization in PDAC in vivo, we will use laser capture microdissection and quantitative polymerase chain reaction (PCR) to assay in the same cancer cells the levels of expression of all four members of the EGFR family. If we exclude gene amplification as a mechanism for in vivo overexpression, we will confirm that our cultured cell lines overexpress these receptors as a result of enhanced transcription in nuclear-run-on studies. We will then characterize their transcriptional control elements in order to develop second generation viral vectors that are preferentially targeted to pancreatic cancer cells.