Mechanisms of SAG Inhibition of Carcinogenesis & Apoptosis

SAG抑制癌变的机制

• 批准号: 7145233
• 负责人: YI SUN
• 金额: $ 26.97万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7145233
• 关键词: antioxidants; apoptosis; carcinogenesis; ligase; role; skin; ubiquitin

DESCRIPTION (provided by applicant): Antioxidants have been previously shown to inhibit skin carcinogenesis induced by tumor initiator/promoter, DMBA/TPA, or UV irradiation, the role of protein ubiquitination and degradation in multistage carcinogenesis is, however, largely unknown. Our long-range goal is to achieve chemo-prevention of skin carcinogenesis through induction of a critical molecule whose expression inhibits such a process. This critical molecule, SAG (Sensitive to Apoptosis Gene) or Rbx2/ROC2, is a cysteine-rich protein and a RING component of SCF (Skp1, Cullins, F-box proteins), possibly of OCX (DDB1/Cul4A/X-box) E3 ubiquitin ligases. We cloned SAG and found that SAG is a redox inducible antioxidant and an E3 ubiquitin ligase. When over-expressed, SAG inhibits apoptosis induced by redox and hypoxia both in vitro and in vivo. The objective of this application is to define an inhibitory role of SAG in skin carcinogenesis and to elucidate its mechanism of action, using a JB6 epidermal cell culture in vitro model and a K14 driven SAG transgenic mouse in vivo model. The central hypothesis is that tumor promoter TPA or carcinogen UV induces AP-1, whereas UV induces p53. Both AP-1 and p53 transactivate SAG expression through a direct binding to their respective consensus elements in the SAG promoter. Upon induction, SAG scavenges ROS or complexes with other components of SCF/DCX E3 ubiquitin ligases to ubiquitinate and degrade c-Jun and cyclin D1, thus protecting epidermal cells from DMBA/TPA- or UV-induced carcinogenesis. The specific a/msto test the hypothesis are 1) to elucidate the mechanism of SAG induction by TPA and UV through transcriptional activation by AP-1 and p53; 2) to define an inhibitory role of SAG in TPA-induced tumor promotion and in UV-induced apoptosis in JB6 epidermal cells; 3) to elucidate mechanism of SAG action as an antioxidant and an E3 ubiquitin ligase; 4) to use SAG transgenic mice to determine the extent to which SAG expression inhibits in vivo skin carcinogenesis induced by DMBA/TPA or UV. Through this research, we will demonstrate that SAG is a novel inhibitor of skin carcinogenesis and that both its antioxidant and E3 ligase activities contribute to such an inhibition. The cancer resistant SAG mice generated here can provide validation of molecular targets (such as AP-1) that when hit, function to prevent carcinogenesis. Furthermore, we will provide a molecular basis for future screening of drugs that may act as chemo-prevention agents via SAG induction.

描述（由申请人提供）：抗氧化剂先前已被证明可以抑制由肿瘤引发剂/促进剂、DMBA/TPA或紫外线照射诱导的皮肤癌发生，然而，蛋白质泛素化和降解在多阶段癌发生中的作用很大程度上是未知的。我们的长期目标是通过诱导一种关键分子的表达来抑制这一过程，从而实现皮肤癌的化学预防。这种关键分子，SAG（细胞凋亡敏感基因）或 Rbx2/ROC2，是一种富含半胱氨酸的蛋白，也是 SCF（Skp1、Cullins、F-box 蛋白）的 RING 成分，可能是 OCX (DDB1/Cul4A/X-box) E3 泛素连接酶的 RING 成分。我们克隆了SAG，发现SAG是一种氧化还原诱导型抗氧化剂，也是一种E3泛素连接酶。当过表达时，SAG 会在体外和体内抑制氧化还原和缺氧诱导的细胞凋亡。本申请的目的是使用 JB6 表皮细胞培养体外模型和 K14 驱动的 SAG 转基因小鼠体内模型来确定 SAG 在皮肤癌发生中的抑制作用并阐明其作用机制。中心假设是肿瘤促进剂 TPA 或致癌物 UV 诱导 AP-1，而 UV 诱导 p53。 AP-1 和 p53 都通过直接结合到 SAG 启动子中各自的共有元件来反式激活 SAG 表达。诱导后，SAG 清除 ROS 或与 SCF/DCX E3 泛素连接酶其他成分的复合物，泛素化并降解 c-Jun 和细胞周期蛋白 D1，从而保护表皮细胞免受 DMBA/TPA 或 UV 诱导的癌变。检验假设的具体a/msto是1）阐明TPA和UV通过AP-1和p53转录激活诱导SAG的机制； 2) 确定SAG在TPA诱导的肿瘤促进和UV诱导的JB6表皮细胞凋亡中的抑制作用； 3) 阐明SAG作为抗氧化剂和E3泛素连接酶的作用机制； 4)利用SAG转基因小鼠测定SAG表达抑制DMBA/TPA或UV诱导的体内皮肤癌发生的程度。通过这项研究，我们将证明 SAG 是一种新型的皮肤癌抑制剂，其抗氧化剂和 E3 连接酶活性都有助于这种抑制作用。这里产生的抗癌 SAG 小鼠可以验证分子靶标（例如 AP-1），当受到攻击时，这些分子靶标可以起到预防癌变的作用。此外，我们将为未来筛选可通过 SAG 诱导充当化学预防剂的药物提供分子基础。