Mechanisms of SAG Inhibition of Carcinogenesis & Apoptosis

描述（由申请人提供）：抗氧化剂先前已被证明可抑制由肿瘤启动剂/启动子、DMBA/TPA或紫外线照射诱导的皮肤癌变，然而，蛋白质泛素化和降解在多阶段癌变中的作用在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皮肤癌的发生，从而实现化学预防皮肤癌的发生。这个关键分子SAG （Sensitive to Apoptosis Gene）或Rbx2/ROC2是一种富含半胱氨酸的蛋白，是SCF （Skp1, Cullins， F-box蛋白）的RING组分，可能是OCX (DDB1/Cul4A/X-box) E3泛素连接酶的组分。我们克隆了SAG，发现SAG是一种氧化还原诱导抗氧化剂和E3泛素连接酶。当SAG过表达时，在体内和体外均可抑制氧化还原和缺氧诱导的细胞凋亡。本应用的目的是通过体外JB6表皮细胞培养模型和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。中心假设是肿瘤启动子TPA或致癌物UV诱导AP-1，而UV诱导p53。AP-1和p53都通过直接结合各自在SAG启动子中的共识元件来反激活SAG表达。经诱导后，SAG清除ROS或与SCF/DCX E3泛素连接酶的其他组分复合物，泛素化并降解c-Jun和cyclin D1，从而保护表皮细胞免受DMBA/TPA或uv诱导的癌变。验证该假设的具体a/ms为：1)阐明TPA和UV通过AP-1和p53的转录激活诱导SAG的机制；2)明确SAG在tpa诱导的JB6表皮细胞肿瘤促进和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，确定SAG表达对DMBA/TPA或UV诱导的体内皮肤癌变的抑制程度。通过这项研究，我们将证明SAG是一种新的皮肤癌抑制剂，其抗氧化和E3连接酶活性都有助于这种抑制。在这里产生的抗癌SAG小鼠可以验证分子靶点（如AP-1），当被击中时，可以起到防止致癌的作用。此外，我们将为未来筛选可能通过SAG诱导作为化学预防剂的药物提供分子基础。

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.