Role of Skp2 and Skp2B in breast cancer

描述(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节事件，包括细胞周期和细胞存活与细胞死亡决定。我们最近发现了一种新的SCF泛素连接酶F-box蛋白Skp2，命名为Skp2B，它在调控乳腺肿瘤发生中的作用。 Skp2是一种核蛋白，在泛素化的底物和SCFSkp2泛素连接酶复合体的核心之间扮演着适配器的角色。Skp2在多种癌症类型中过度表达，并与癌症进展有关。我们已经分离到Skp2B，一种定位于细胞质的剪接变体。我们发现Skp2B并不是Skp2的显性否定形式。此外，我们还发现Skp2B在大量的原发乳腺癌中过表达，并且在MMTV-Skp2B转基因模型中过表达导致乳腺癌，并加速人乳腺癌裸鼠移植瘤的生长。此外，使用同时识别Skp2和Skp2B的抗体对Skp2进行的免疫组织化学分析显示，在大量乳腺癌样本中，Skp2染色是细胞质的，这表明Skp2B而不是Skp2在这些癌症中过表达。在功能方面，我们发现Skp2B定位于线粒体，我们通过酵母双杂交筛选分离到了与Skp2B特异相互作用的线粒体蛋白的一个子集。特别令人感兴趣的是线粒体伴侣BAP37，它面对膜间隙(IMS)，其中驻留着几个促凋亡蛋白。重要的是，我们发现BAP37与泛素化蛋白有关，Skp2B促进错误折叠的IMS蛋白的泛素化，shRNA抑制Skp2B诱导自发凋亡。这些新的发现为我们的假设提供了基础：Skp2B与BAP37的相互作用奠定了质量控制的基础，该质量控制监测错误折叠的IMS蛋白的存在，并通过泛素化将其消除。此外，Skp2B的过表达可以保护乳腺癌细胞免受IMS应激诱导的细胞凋亡。我们将使用我们的MMTV-Skp2B转基因小鼠模型来验证我们的假设，通过设计一个错误折叠的IMS蛋白并测试其被Skp2B泛化的能力，最后我们将分离与Skp2B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.