Role of Skp2 and Skp2B in breast cancer

描述（由申请人提供）：泛素修饰越来越被认为是影响癌症发展的基本细胞生物学过程中的关键调控事件，包括细胞周期和细胞存活与细胞死亡决定。本申请的重点是SCF泛素连接酶F-box蛋白Skp 2，称为Skp 2B，我们最近发现，在乳腺肿瘤发生的调节中的一个新的亚型的作用。 Skp 2是一种核蛋白，作为泛素化底物和SCF Skp 2泛素连接酶复合物核心之间的衔接子。Skp 2在多种癌症类型中过表达，并与癌症进展有关。我们已经分离出Skp 2B，一种定位于细胞质的剪接变体。我们发现Skp 2B并不作为Skp 2的显性负性形式。此外，我们发现Skp 2B在大量原发性乳腺癌中过表达，并且其过表达导致MMTV-Skp 2B转基因模型中的乳腺癌和裸鼠中人乳腺癌异种移植物生长的加速。此外，使用识别Skp 2和Skp 2B两者的抗体对Skp 2进行免疫组织化学分析揭示，在大量乳腺癌样品中，Skp 2染色是细胞质的，这表明Skp 2B而不是Skp 2在这些癌症中过表达。就其功能而言，我们发现Skp 2B定位于线粒体，并且我们已经通过酵母双杂交筛选分离出与Skp 2B特异性相互作用的线粒体蛋白质的子集。特别令人感兴趣的是线粒体伴侣蛋白BAP 37，其面向膜间空间（IMS），其中存在几种促凋亡蛋白。重要的是，我们发现BAP 37与泛素化蛋白相关，Skp 2B促进错误折叠的IMS蛋白的泛素化，并且通过shRNA抑制Skp 2B诱导自发凋亡。这些新的发现为我们的假设提供了基础：Skp 2B与BAP 37的相互作用是质量控制的基础，该质量控制监测错误折叠的IMS蛋白的存在，并通过泛素化靶向其消除。此外，Skp 2B过表达保护乳腺癌细胞免受IMS应激诱导的凋亡。我们将使用我们的MMTV-Skp 2B转基因小鼠模型来验证我们的假设，通过工程化错误折叠的IMS蛋白并测试其被Skp 2B泛素化的能力，最后我们将分离与Skp 2B相关的泛素连接酶。

DESCRIPTION (provided by applicant): Ubiquitin modifications are increasingly recognized as key regulatory events in basic cell biology processes that impact the development of cancer, including cell cycle and cell survival versus cell death decisions. This application focuses on the role of a new isoform of the SCF ubiquitin ligase F-box protein Skp2, termed Skp2B, which we recently identified, in the regulation of mammary tumorigenesis. Skp2 is a nuclear protein that acts as an adaptor between substrates for ubiquitination and the core of the SCFSkp2 ubiquitin ligase complex. Skp2 is overexpressed in a variety of cancer types and has been linked to cancer progression. We have isolated Skp2B, a splice variant that localizes to the cytoplasm. We found that Skp2B does not act as a dominant negative form of Skp2. Further, we found that Skp2B is overexpressed in a large number of primary breast cancers, and that its overexpression leads to mammary carcinoma in an MMTV-Skp2B transgenic model and acceleration of the growth of human breast cancer xenografts in nude mice. Further, immunohistochemical analysis of Skp2 using antibodies that recognize both Skp2 and Skp2B, revealed that in a large number of breast cancer samples, Skp2 staining is cytoplasmic suggesting that Skp2B rather than Skp2 is overexpressed in these cancers. In terms of its function, we found that Skp2B localizes to the mitochondria and we have isolated a subset of mitochondria proteins that interact specifically with Skp2B by a yeast two-hybrid screen. Of particular interest is the mitochondrial chaperone BAP37 that faces the inter membrane space (IMS), where several pro-apoptotic proteins reside. Importantly, we found that BAP37 associates with ubiquitinated proteins that Skp2B promotes the ubiquitination of a misfolded IMS protein and that inhibition of Skp2B by shRNA induces spontaneous apoptosis. These novel findings provide the foundation of our hypothesis that: Skp2B interaction with BAP37 underlies a quality control that monitors the presence of misfolded IMS proteins and targets their elimination via ubiquitination. Further, that Skp2B overexpression protects breast cancer cells from IMS stress-induced apoptosis. We will test our hypothesis using our MMTV-Skp2B transgenic mice model, by engineering a misfolded IMS protein and test its ability to be ubiqutinated by Skp2B, finally we will isolate the ubiquitin ligase in association with Skp2B.