The role of RalA in pancreatic tumorigenesis

胰腺癌的预后很差，很大程度上是由于目前的治疗方法无效。 养生法。我们的长期目标是确定导致胰腺癌发展的关键因素，以便 他们可以成为新的治疗策略的目标。我们的假设是两个K-RAS对Rala的激活 而Aurora-A激酶是胰腺肿瘤发生过程中的关键步骤。这一假设基于以下几点：1) 几乎所有胰腺癌都有癌基因K-RAS的激活突变，K-RAS对肿瘤的发生至关重要， 而这种致癌信号在很大程度上是由人类的Ralgef途径介导的；2)转化 Ralgef效应子Rala对胰腺癌细胞系的转化和致瘤生长都是至关重要的； 3)被Aurora-A激酶磷酸化的Rala丝氨酸194是Ralgef转化所必需的。 依赖的人类癌细胞，以及4)Aurora-A抑制减少胰腺的致瘤生长 癌细胞系。我们建议评估Rala和Aurora-A在胰腺肿瘤发生中的作用： 具体目标1：确定Rala的缺失是否抑制K-ras诱导的小鼠胰腺肿瘤的发生。我们会 评价有条件表达致癌基因的转基因小鼠的自发性胰腺肿瘤发生 胰腺K-ras等位基因，但不存在Rala等位基因。 具体目标2：确定Rala是否通过其已知的效应器RalBPI、Exo84或Sec5发挥作用 在胰腺癌的发生发展中起中介作用。我们将a)对候选人进行功能损失分析 Rala下游效应器RalBPI、Exo84或Sec5，并确定对转换和 胰腺癌细胞系的致瘤生长，以及b)决定Rala S194的磷酸化 更改RalBPI、Exo84或Sec5的Rala的绑定亲和力。 特定目标3：确定Rala S194的Aurora-A磷酸化是否促进胰腺癌 成长。我们将a)确定Rala S194是否需要被Aurora-A磷酸化才能转化 和胰腺癌细胞系的致瘤生长，b)确定抑制Aurora-A是否需要RALA S194以抑制胰腺癌细胞的生长，以及c)确定Rala S194是否磷酸化 导致Rala在胰腺癌细胞中的细胞内重新定位。

Pancreatic cancer has a dismal prognosis due in large part to the ineffectiveness of current treatment regimens. Our long-term goal is to identify key factors underlying pancreatic cancer development so that they can be targeted for new therapeutic strategies. Our hypothesis is that activation of RalA by both K-Ras and Aurora-A kinase is a key step in pancreatic tumorigenesis. This hypothesis is based on the following: 1) Nearly all pancreatic cancers have an activating mutation in the oncogene K-Ras critical for tumorigenesis, and this oncogenic signal is mediated in large part by the RalGEF pathway in humans; 2) the transforming RalGEF effector RalA is crucial for both transformed and tumorigenic growth of pancreatic cancer cell lines; 3) RalA serine 194, which is phosphorylated by Aurora-A kinase, is required for transformation of RalGEF- dependent human cancer cells, and 4) Aurora-A inhibition decreases tumorigenic growth of pancreatic cancer cell lines. We propose to evaluate the roles of RalA and Aurora-A in pancreatic tumorigenesis: Specific Aim 1: Determine if loss of RalA inhibits K-ras-driven pancreatic tumorigenesis in mice. We will evaluate spontaneous pancreatic tumorigenesis in transgenic mice which conditionally express anoncogenic K-ras allele but not RalA in the pancreas. Specific Aim 2: Determine whether RalA functions through its known effectors RalBPI, Exo84, or Sec5 to mediate the development of pancreatic cancer. We will a) perform loss-of-function analysis of the candidate RalA downstream effectors RalBPI, Exo84, or Sec5, and determine the effect on the transformation and tumorigenic growth of pancreatic cancer cell lines, and b) determine whether phosphorylation of RalA S194 alters the binding affinities of RalA for RalBPI, Exo84, or Sec5. Specific Aim 3: Determine whether Aurora-A phosphorylation of RalA S194 enhances pancreaticcancer growth. We will a) establish whether phosphorylation of RalA S194 by Aurora-A is required for transformed and tumorigenic growth in pancreatic cancer cell lines, b) establish whether Aurora-A inhibition requires RalA S194 to decrease growth of pancreatic cancer cells, and c) determine whether RalA S194 phosphorylation causes intracellular relocalization of RalA in pancreatic cancer cells.