Mechanisms of SAG Inhibition of Carcinogenesis & Apoptosis

说明(申请人提供)：抗氧化剂已被证明可以抑制由肿瘤启动剂/促进剂、DMBA/TPA或紫外线辐射诱导的皮肤癌变，然而，蛋白质泛素化和降解在多阶段癌变中的作用尚不清楚。我们的长期目标是通过诱导一种关键分子的表达来实现皮肤癌的化学预防，该分子的表达抑制了这一过程。这个关键分子SAG或Rbx2/ROC2是一种富含半胱氨酸的蛋白，是SCF(Skp1、Cullins、F-box蛋白)的环成分，可能是OCX(DDB1/Cul4A/X-box)E3泛素连接酶的环成分。我们克隆了SAG，发现SAG是一种氧化还原诱导型抗氧化剂和E3泛素连接酶。当SAG过表达时，在体内外均可抑制氧化还原和低氧诱导的细胞凋亡。本研究的目的是利用JB6表皮细胞体外培养模型和K14驱动的SAG转基因小鼠体内模型，明确SAG在皮肤癌变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或致癌物紫外线诱导AP-1，而紫外线诱导p53。AP-1和P53通过直接结合各自的SAG启动子中的共同元件反式激活SAG的表达。在诱导后，SAG清除ROS或与SCF/DCX E3泛素连接酶其他成分的复合体，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或紫外线诱导的致癌。验证假设的特异性a/mst为1)通过AP-1和P53的转录激活，阐明TPA和UV诱导SAG的机制；2)确定SAG在TPA诱导的促肿瘤和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)使用SAG转基因小鼠，确定SAG在体内抑制DMBA/TPA或UV诱导的皮肤癌变的程度。通过这项研究，我们将证明SAG是一种新的皮肤癌变抑制剂，它的抗氧化剂和E3连接酶活性都参与了这种抑制作用。这里产生的抗癌SAG小鼠可以提供分子靶点(如AP-1)的验证，当击中这些靶点时，就会起到预防癌症发生的作用。此外，我们将为未来通过SAG诱导筛选可能起化学预防作用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.