SAG E3 ubiquitin ligase in regulation of transformation and carcinogenesis

描述（由申请人提供）：SAG (Sensitive to Apoptosis Gene)，也被称为RBX2/ROC2，是SCF （Skp1, Cullins， F-box蛋白）E3泛素连接酶的应激响应环组分，是其活性所必需的。我们强有力的初步数据和已发表的结果表明：1)Sag是小鼠胚胎发育过程中生长所必需的基因，无Sag的mef和ES细胞分别在体外培养和裸鼠培养时生长迟缓；2) SAG转基因表达通过抑制细胞凋亡促进DMBA/ tpa诱导的皮肤乳头瘤的生长；3) SAG在多种人类癌症中过度表达，尤其是肺癌；4)SAG siRNA沉默通过诱导细胞凋亡抑制癌细胞生长。然而，Sag是否为永生化、转化或致癌所必需，从而作为化学预防和治疗的有效癌症靶点，尚未得到研究。本应用程序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以及肺和皮肤组织癌变中的作用。核心假设是，Sag在过表达时，通过促进肿瘤抑制和凋亡诱导底物（如IkB、Nf1、p27和Noxa）的降解，从而激活NFkB和Ras通路，从而促进细胞生长和抑制细胞凋亡。另一方面，Sag通过基因缺失或小分子抑制剂MLN4924灭活后，会引起这些底物的积累，使NFkB和Ras通路失活，从而抑制增殖和致癌作用。本文提出了三个具体目的来阐明Sag在E1A/Hras诱导小鼠胚胎成纤维细胞永生化和转化中的作用；2) KrasG12D诱导的肺癌，3)DMBA/TPA诱导的皮肤癌。影响：我们的工作是高度创新的，具有重要的翻译价值，通过验证SAG E3泛素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MLN4924的未来开发提供一些概念验证证据，MLN4924是一种新发现的SCF E3连接酶的小分子抑制剂，作为一类新的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.