SAG E3 ubiquitin ligase in regulation of transformation and carcinogenesis

描述（由申请人提供）：SAG（细胞凋亡敏感基因），也称为RBX 2/ROC 2，是SCF（Skp 1，Cullins，F-box蛋白）E3泛素连接酶的应激响应性RING组分，是其活性所需。我们的初步研究和已发表的研究结果表明：1）Sag基因是小鼠胚胎发育过程中生长所必需的基因，Sag基因缺失的MEFs和ES细胞在体外培养和裸鼠体内生长均受到抑制; 2）SAG转基因表达通过抑制细胞凋亡促进DMBA/TPA诱导的皮肤乳头状瘤的生长; 3）SAG在多种人类癌症，特别是肺癌中过表达，和4）SAG siRNA沉默通过诱导细胞凋亡抑制癌细胞生长。然而，是否需要Sag的永生化，转化，或致癌，从而作为一个有效的癌症靶点的化学预防和治疗，还没有检查。本申请的目的是在生理环境下使用组织特异性Sag敲除小鼠模型来研究Sag在原代培养物的永生化和转化中以及在肺和皮肤组织的致癌作用中的作用。中心假设是，Sag在过表达时，通过促进肿瘤抑制和凋亡诱导底物如IkB、Nf 1、p27和Noxa的降解，促进细胞生长并抑制凋亡，导致NFkB和Ras途径的激活。另一方面，通过基因缺失或用小分子抑制剂MLN 4924处理失活后，Sag会导致这些底物蓄积，从而阻断NFkB和Ras途径，从而抑制增殖和致癌作用。本研究拟从三个方面阐明Sag在E1 A/Hras介导的小鼠胚胎成纤维细胞永生化和转化、KrasG 12 D诱导的肺癌发生和DMBA/TPA诱导的皮肤癌发生中的作用。影响：我们的工作具有高度创新性，并具有重大的翻译价值，通过验证SAG E3遍在蛋白连接酶作为癌症化学预防和治疗的有吸引力的靶点，从而为MLN 4924（一种新发现的小分子抑制剂）的未来开发提供了一些概念验证证据。SCF E3连接酶，作为一类新型化学预防和抗癌药物。

DESCRIPTION (provided by applicant): SAG (Sensitive to Apoptosis Gene), also known as RBX2/ROC2, is a stress-responsive RING component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase, required for its activity. Our strong preliminary data as well as published results showed that 1) Sag is a gene essential for growth during mouse embryonic development, and Sag-null MEFs or ES cells are growth retarded when cultured in vitro or grown in nude mice, respectively; 2) SAG transgenic expression accelerates the growth of DMBA/TPA-induced skin papilloma by inhibiting apoptosis; 3) SAG is overexpressed in multiple human cancers, particularly lung cancer, and 4) SAG siRNA silencing suppresses cancer cell growth via inducing apoptosis. However, whether Sag is required for immortalization, transformation, or carcinogenesis, thus serving as a valid cancer target for chemoprevention and therapy, has not been examined. The objectives of this application are to use tissue specific Sag knockout mouse models at physiological settings to study the role of Sag in immortalization and transformation of primary cultures and in carcinogenesis of lung and skin tissues. The central hypothesis is that Sag, upon overexpression, promotes cell growth and inhibits apoptosis via promoting the degradation of tumor suppressive and apoptosis-inducing substrates such as IkB, Nf1, p27, and Noxa, leading to activation of the NFkB and Ras pathways. On the other hand, Sag, upon inactivation by genetic deletion or treatment with small molecule inhibitor MLN4924, causes an accumulation of these substrates to inactivate the NFkB and Ras pathways, resulting in suppression of proliferation and carcinogenesis. Three specific aims are proposed to elucidate the role of Sag in 1) immortalization and transformation of mouse embryonic fibroblasts by E1A/Hras; 2) lung carcinogenesis induced by KrasG12D, and 3) skin carcinogenesis induced by DMBA/TPA. IMPACT: our work is highly innovative and of significant impact with translational value by validating SAG E3 ubiquitin ligase as an attractive target for cancer chemoprevention and therapy, thus providing some proof-of-concept evidence for future development of MLN4924, a newly discovered small molecule inhibitor of SCF E3 ligase, as a novel class of chemoprevention and anticancer agents.