The role of FBXW2 as a novel lung tumor suppressor that cross-talks with oncogenic beta-TrCP and SKP2

描述(申请人提供)：SCF(Skp1-Cullins-F box Protein)E3泛素连接酶是E3连接酶中最大的家族，它促进各种关键调控蛋白的泛素化和降解，从而控制许多重要的生物学过程，如细胞凋亡、细胞周期进展、DNA复制、胚胎发生和肿瘤发生。SCF由4个组分组成，包括支架蛋白cullin-1、接头蛋白Skp1、环蛋白RBX1或RBX2和F-box蛋白。哺乳动物细胞中有69个成员的F-box蛋白是决定SCF E3s特异性的底物识别亚基。虽然已经通过一种特性良好的F-box蛋白？TrCP、FBXW7或Skp2鉴定了多种蛋白质底物，但对于F-box蛋白是否以及如何相互调节知之甚少。我们的初步数据表明：1)致癌F-box蛋白TrCP与一个尚未得到很好鉴定的F-box蛋白FBXW2结合，从而促进其泛素化和降解；2)FBXW2反过来与致癌F-box蛋白Skp2结合，促进其泛素化和降解；3)FBXW2对肺癌细胞具有抑制作用；4)在人非小细胞肺癌组织中发现了一系列功能获得突变；以及5)β-TrCP、FBXW2和Skp2在肺癌组织中的表达存在反向相关，与患者的生存相关。尽管β-TrCP和Skp2是研究较多的具有致癌活性的F-box蛋白，但它们是否以及如何在细胞增殖和存活方面相互调节是完全未知的。本研究的目的是通过体外细胞培养和体内转基因小鼠模型，了解FBXW2如何介导和调节两种已知的致癌F-box蛋白β-TrCP和Skp2的活性，以及FBXW2如何作为一种新的肿瘤抑制因子在肺肿瘤发生中发挥作用，并阐明其作用机制。中心假设是三个F-盒蛋白，β，TrCP-FBXW2-Skp2，形成一个癌基因-抑癌基因-癌基因轴，相互调节，控制细胞增殖和肺肿瘤的发生。我们提出了三个具体的目标来验证我们的中心假设：1)将FBXW2表征为β-TRCP的新底物；2)将FBXW2表征为降解Skp2的新的E3连接酶；以及3)将FBXW2表征为抗肺肿瘤发生的新的肿瘤抑制因子。相关性：这项拟议的研究的成功完成将从机制上阐明三个F-box蛋白如何通过相互靶向降解来调节肺癌细胞的增殖，两个致癌F-box蛋白ç-TrCP和Skp2如何通过肿瘤抑制F-box蛋白FBXW2相互交谈，以及FBXW2如何在肺中作为肿瘤抑制因子，从而阐明肺肿瘤发生的新机制，并为未来肺癌治疗中生物标记物的开发提供有吸引力的靶点。

DESCRIPTION (provided by applicant): SCF (SKP1-Cullins-F box protein) E3 ubiquitin ligase is the largest family of E3 ligases that promote the ubiquitylation and degradation of various key regulatory proteins, thus controlling many important biological processes, such as apoptosis, cell cycle progression, DNA replication, embryogenesis and tumorigenesis. SCF consists of 4 components, including a scaffold protein cullin-1, an adaptor protein SKP1, a RING protein RBX1 or RBX2 and an F-box protein. The F-box protein with 69 members in mammalian cells is the substrate- recognizing subunit that determines the specificity of SCF E3s. While a variety of protein substrates have been identified by one of well-characterized F-box proteins ßTrCP, FBXW7, or SKP2, very little is known as to whether and how F-box proteins regulate each other. Our preliminary data showed that 1) oncogenic F-box protein TrCP binds to yet a poorly characterized F-box protein FBXW2 to promote its ubiquitylation and degradation; 2) FBXW2 in turn binds to oncogenic F-box protein SKP2 to promote its ubiquitylation and degradation; 3) FBXW2 has tumor suppressive function against lung cancer cells; 4) A number of gain-of- function FBXW2 mutations were found in human non-small cell lung carcinoma tissues; and 5) reversed correlation in expression among β-TrCP, FBXW2 and SKP2 exists in lung cancer tissues, which is associated with patient survival. Although β-TrCP and SKP2 are well-studied F-box proteins with oncogenic activity, whether and how they regulate each other with regard to cell proliferation and survival is totally unknown. The objectives of this proposed study is to understand how FBXW2 mediates and modulates the activity of two well-known oncogenic F-box proteins, β-TrCP and SKP2, and how FBXW2 acts as a novel tumor suppressor in lung tumorigenesis using both in vitro cell culture and in vivo genetically modified mouse models with elucidation of its mechanism of action. The central hypothesis is that three F- box proteins, βTrCP-FBXW2-SKP2 form an oncogene-tumor suppressor-oncogene axis that regulates each other to control cell proliferation and lung tumorigenesis. Three specific aims are proposed to test our central hypothesis by 1) characterizing FBXW2 as a novel substrate of β-TRCP; 2) characterizing FBXW2 as a novel E3 ligase for SKP2 degradation; and 3) characterizing Fbxw2 as a novel tumor suppressor against lung tumorigenesis. Relevance: Successful completion of this proposed study will mechanistically elucidate how three F-box proteins regulate proliferation of lung cancer cells by targeting each other for degradation, and how two oncogenic F-box proteins ß-TrCP and SKP2 talk with each other via a tumor suppressive F-box protein FBXW2, and how FBXW2 acts as a tumor suppressor in the lung, thus elucidating a novel mechanism of lung tumorigenesis and providing attractive targets for future biomarker development in lung cancer management.