NRDP1 PROTEIN DEGRADATION PATHWAY IN MAMMARY TUMOR PROGRESSION

描述(申请人提供)：生长因子受体酪氨酸激酶ErbB家族成员的异常过度表达和伴随的激活在促进各种实体肿瘤类型的生长和进展中起着关键作用。这项研究的长期目标是了解一种新的蛋白质降解途径在调节ErbB3诱导的乳腺肿瘤发生和发展中的作用。该途径的中心成分是一种名为Nrdp1的环指E3泛素连接酶，它介导ErbB3受体的泛素化，从而促进其运输到降解的细胞室。在ErbB2诱导的过表达内源性ErbB3的转基因小鼠的乳腺肿瘤中，Nrdp1蛋白总是转录后被抑制，并且几乎60%的乳腺癌患者的肿瘤中Nrdp1蛋白丢失，这与Nrdp1缺失促进ErbB3蛋白在肿瘤中过表达的模型一致。然而，最近的证据表明，Nrdp1及其相关的途径组件在细胞调控中发挥着更广泛的作用。指导这项研究的假设是，Nrdp1通过多条途径维持乳腺上皮细胞的完整性，转录后Nrdp1蛋白的缺失消除了乳腺肿瘤启动的障碍。关于Nrdp1参与乳腺上皮完整性的三个关键问题将被解决。1)Nrdp1途径相关的脱泛素酶Otub1在肿瘤细胞中介导Nrdp1转录后缺失的作用将通过病毒介导的培养细胞中的过表达和敲除来评估。此外，Otub1在乳腺中的可诱导转基因过表达将被用来评估其在乳腺发育和ErbB2诱导的肿瘤发生中的作用。2)利用病毒介导的过表达和基因敲除方法，结合生化分析和免疫荧光细胞结构分析，探讨Nrdp1通路组件在建立和维持培养上皮细胞的极性和结构中的作用。3)Nrdp1基因在抑制ErbB2诱导的肿瘤发病中的作用将通过条件基因敲除和移植的方法来检验。总之，拟议的研究可能揭示该途径在乳腺肿瘤发展中的新活性，并强调其在乳腺癌抑制中的作用。

DESCRIPTION (provided by applicant): The aberrant over expression and concomitant activation of members of the ErbB family of growth factor receptor tyrosine kinases plays key roles in promoting the growth and progression of a variety of solid tumor types. The long term objective of the proposed studies is to understand the role of a novel protein degradation pathway in regulating ErbB3-induced mammary tumor initiation and progression. The central component of the pathway is a RING finger E3 ubiquitin ligase called Nrdp1 that mediates the ubiquitination of the ErbB3 receptor, thereby promoting its trafficking to degradative cellular compartments. Nrdp1 protein is invariably post-transcription ally suppressed in ErbB2-induced mammary tumors from transgenic mice that over express endogenous ErbB3, and Nrdp1 protein is lost in tumors from almost 60% of breast cancer patients, consistent with a model whereby Nrdp1 loss facilitates ErbB3 protein over expression in tumors. However, recent evidence points to a broader role for Nrdp1 and its associated pathway components in cellular regulation. The hypothesis guiding the proposed studies is that Nrdp1 acts through multiple pathways to maintain mammary epithelial cell integrity, and that the post-transcriptional loss of Nrdp1 protein removes a barrier to mammary tumor initiation. Three critical issues concerning Nrdp1 involvement in mammary epithelial integrity will be addressed. 1) The role of the Nrdp1 pathway-associated deubiquitinating enzyme Otub1 in mediating Nrdp1 post-transcriptional loss in tumor cells will be assessed by viral-mediated over expression and knockdown in cultured cells. In addition, inducible transgenic over expression of Otub1 in the mammary gland will be used to assess its involvement in mammary gland development and ErbB2-induced tumorigenesis. 2) The role of Nrdp1 pathway components in establishing and maintaining the polarity and architecture of cultured epithelial cells will be explored using viral-mediated over expression and knockdown approaches coupled with biochemical assays and immunofluoresecence-based cell structure assays. 3) The role of the Nrdp1 gene in suppressing the onset of ErbB2-induced tumors will be examined using conditional knockout and transplantation approaches. Together, the proposed studies could uncover novel activities for this pathway in mammary tumor development, and underscore its role in mammary tumor suppression.