Regulation of Stat Function in Breast Cancer

描述（由申请方提供）：Stat家族转录因子是乳腺组织生长和分化所必需的。在乳腺组织中，Stat激活的一个重要手段是神经内分泌激素催乳素（PRL）与其受体PRLr的结合。PRL/PRLr结合导致受体相关信号传导网络的激活，例如Jak/Stat途径以及PRL与脯氨酰异构酶亲环蛋白B（CypB）之间复合物的内吞作用和核逆向转运。核内PRL/CypB复合物调节Stat 5介导的基因表达和DNA结合。使用多种方法，包括酵母双杂交和转录因子阵列分析，我们已经证明，PRL/CypB复合物调节Stat 5与多种核内蛋白的相互作用，包括活化的Stat的肽抑制剂PIAS 3，小泛素相关的修饰物SUMO 2，以及转录因子和原癌基因c-Myb。这是该提议的中心假设，Stat 5与这些蛋白质的时间和空间相互作用，如由PRL/CypB复合物调节的，调节Stat 5功能。这一假设将在体外和体内使用乳腺癌和PRL反应细胞系通过三个特定目的进行测试。首先，Stat 5/PIAS 3相互作用的结构基础和这种相互作用的功能意义将使用互补诱变/过表达和敲低策略进行映射。第二，Stat 5 summolyation的功能后果将通过细胞和生物化学方法进行评估。第三，Stat 5和共激活物/阻遏物（如c-Myb）之间的关联将通过靶向诱变、小干扰RNA（SiRNA）和基于染色质免疫沉淀（ChIP）的方法来评估。鉴于我们的初步证据表明，操纵这些相互作用的结果在Stat 5介导的基因表达和乳腺癌细胞的生存的改变，我们提出的研究是直接相关的病理生理学的更好的理解：人类乳腺癌，因此，可能会提供新的治疗策略，旨在修改Stat功能在这种疾病的基础。

DESCRIPTION (provided by applicant): The Stat family of transcription factors is necessary for the growth and differentiation of mammary tissues. In mammary tissues, a significant means of Stat activation is the binding of the neuroendocrine hormone prolactin (PRL) to its receptor, the PRLr. PRL/PRLr binding results in the activation of receptor-associated signaling networks, such as the Jak/Stat pathway and the endocytosis and nuclear retrotransport of a complex between PRL and the prolyl isomerase cyclophilin B (CypB). The intranuclear PRL/CypB complex regulates Stat5-mediated gene expression and DNA binding. Using multiple approaches including yeast two-hybrid and transcription factor array analysis, we have demonstrated that the PRL/CypB complex regulates the interaction of Stat5 with multiple intranuclear proteins including the peptide inhibitor of activated Stat, PIAS3, the small ubiquitin-related modifier, SUMO2, and the transcription factor and proto-oncogene, c-Myb. It is the central hypothesis of this proposal that the temporal and spatial interaction of Stat5 with these proteins, as modulated by the PRL/CypB complex, regulates Stat5 function. This hypothesis will be tested by three specific aims using breast cancer and PRL-responsive cell lines both in vitro and in vivo. First, the structural basis for Stat5/PIAS3 interaction and the functional significance of this interaction will be mapped using complementary mutagenesis/overexpression and knockdown strategies. Second, the functional consequences of Stat5 sumolyation will be evaluated by cellular and biochemical approaches. Third, the association between Stat5 and co-activators/repressors, such as c-Myb will be assessed by targeted mutagenesis, small-interfering RNA (SiRNA), and chromatin immunoprecipitation (ChlP)-based methodologies. Given that our preliminary evidence indicates that the manipulation of these interactions results in the alteration of Stat5-mediated gene expression and the survival of breast cancer cells, our proposed studies are directly relevant to a better understanding of the pathophysiology of: human breast cancer, and as such, may provide the basis for novel therapeutic strategies aimed at modifying Stat function in this disease.