SAG E3 ubiquitin ligase in angiogenesis & carcinogenesis

SAG（Sensitive to Apoptosis Gone），也称为RBX 2 IROC 2或RNF 7（RING finger protein 7），是SCF（Skp 1，Cullins，F-box proteins）E3泛素连接酶的应激反应组分。我们以前的工作表明，SAG在应激条件下促进细胞增殖并抑制细胞凋亡。然而，SAG在调节血管生成和癌发生中的作用在很大程度上是未知的。我们的长期目标是通过靶向SAG抑制血管生成和肿瘤发生来实现癌症治疗。我们强有力的初步数据显示，SAG敲除导致E10.5 -12.5的小鼠胚胎死亡，这主要是由于血管发生和血管生成的失败。来源于SAG缺失的胚胎干（ES）细胞的类胚体不能分化为内皮细胞以形成血岛内部，这与VEGF诱导和RAS/ERK激活的失败有关。来源于SAG缺失ES细胞的畸胎瘤体积较小，血管密度降低，增殖速率降低。我们还发现SAG的转基因表达加速了化学致癌物诱导的小鼠皮肤肿瘤的生长，而通过siRNA敲低SAG抑制了体外和体内人类癌细胞的生长。本申请的目的是1）确定SAG在血管生成中的作用和2）使用小鼠ES细胞和人癌细胞阐明其作用机制。中心假设是SAG通过诱导1 KB的降解来激活血管生成， NF-κ B和神经纤维蛋白（NF-1基因产物）降解以激活RAS。SAG缺失导致1）IKBa积累成NF κ B，和2）神经纤维蛋白积累成RAS，导致血管生成的抑制。检验该假设的具体目的是：1）确定SAG在小鼠ES细胞和人癌细胞中调节血管生成的作用，和2）确定SAG调节血管内皮细胞分化的作用机制。 胚状体我们预期，成功完成这项拟议的研究将证明SAG的阻断通过积聚的1 KB灭活NF κ B和积聚的神经纤维蛋白灭活RAS来抑制血管生成。因此，通过siRNA沉默或小分子抑制剂靶向癌细胞中的SAG将是有效的抗血管生成疗法。

SAG (Sensitive to Apoptosis Gone), also known as RBX2IROC2, or RNF7 (RING finger protein 7), is a stress-responsive component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase. Our previous work has shown that SAG promotes cell proliferation and Inhibits apoptosis under stressed conditions. The role of SAG in regulation of angiogenesis and carcinogenesis is, however, largely unknown. Our long-range goal is to achieve cancer therapy through inhibition of angiogenesis and tumorigenesis by targeting SAG. Our strong preliminary data showed that SAG knockout causes mouse embryonic lethality at E1O.5-12.5, largely due to the failure in vasculogenesis and angiogenesis. Embryoid bodies derived from SAG-null embryonic stem (ES) cells are unable to differentiate into endothelial cells to form the interior of blood islands, which is associated with the failure in VEGF induction and in RAS/ERK activation. The teratomas derived from SAG-null ES cells are smaller in size with reduced density of blood vessels and reduced rate of proliferation. We also found that transgenic expression of SAG accelerates the growth of mouse skin tumors induced by chemical carcinogens, whereas SAG knock-down by siRNA inhibits the growth of human cancer cells both in vitro and in vivo. The objectives of this application are 1) to define the role of SAG in angiogenesis and 2) to elucidate its mechanism of action, using mouse ES cells arid human cancer cells. The central hypothesis is that SAG promotes angiogenesis by inducing degradation of 1KB to activate NFKB and degradation of neurofibromin, a NF-1 gene product, to activate RAS. SAG deletion causes the accumulation of 1) lKBa to inactivate NFKB, and 2) neurofibromin to inactivate RAS, resulting in inhibition of angiogenesis. The specific aims to test the hypothesis are 1) to define the role of SAG in regulation of angiogenesis in mouse ES cells and in human cancer cells and 2) to define the mechanism of action by which SAG regulates endothelial differentiation of embryoid bodies. We expect that successful completion of this proposed study will demonstrate that blockade of SAG inhibits angiogenesis through NFKB inactivation by accumulated 1KB and RAS inactivation by accumulated neurofibromin. Targeting SAG in cancer cells, via siRNA silencing or small molecule inhibitors, would, therefore, be a valid antianglogenesis therapy.