Regulation of Stat Function in Breast Cancer

描述(申请人提供)：Stat转录因子家族是乳腺组织生长和分化所必需的。在乳腺组织中，STAT激活的一个重要途径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网络的激活，如Jak/Stat途径，以及PRL和Pro异构酶亲环素B(CypB)之间的复合体的内吞和核反向转运。核内PRL/CypB复合体调节Stat5介导的基因表达和DNA结合。利用酵母双杂交和转录因子阵列分析等多种方法，我们已经证明了PRL/CypB复合体调节Stat5与多种核内蛋白的相互作用，包括激活STAT的多肽抑制物PIAS3，小泛素相关修饰物SUMO2，以及转录因子和原癌基因c-Myb。这一假设的中心假设是，在PRL/CypB复合体的调控下，Stat5与这些蛋白的时空相互作用调节着Stat5的功能。这一假设将通过三个特定的目标在体外和体内通过乳腺癌和PRL反应细胞株进行验证。首先，将使用互补突变/过表达和敲除策略来定位Stat5/PIAS3相互作用的结构基础和这种相互作用的功能意义。其次，将通过细胞和生化方法评估Stat5总和的功能后果。第三，将通过靶向突变、小干扰RNA(SiRNA)和染色质免疫沉淀(ChlP)的方法来评估Stat5和共激活/抑制因子之间的关联，如c-Myb。鉴于我们的初步证据表明，这些相互作用的操纵导致STAT5介导的基因表达的改变和乳腺癌细胞的存活，我们拟议的研究直接与更好地理解人类乳腺癌的病理生理相关，因此，可能为旨在改变这种疾病的STAT功能的新的治疗策略提供基础。

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.