SAG E3 ubiquitin ligase in regulation of transformation and carcinogenesis

描述(申请人提供)：SAG(凋亡敏感基因)，也被称为RBX2/ROC2，是SCF(Skp1，Cullins，F-box蛋白)E3泛素连接酶的一个应激反应环成分，是其活性所必需的。我们强大的初步数据以及已发表的结果表明：1)SAG是小鼠胚胎发育过程中生长所必需的基因，而SAG缺失的MEF或ES细胞在体外培养或在裸鼠体内生长时分别具有生长抑制作用；2)SAG转基因表达通过抑制细胞凋亡而加速DMBA/TPA诱导的皮肤乳头状瘤的生长；3)SAG在多种人类肿瘤，尤其是肺癌中过表达；4)SAG siRNA沉默通过诱导凋亡抑制癌细胞生长。然而，SAG是否是永生化、转化或致癌所必需的，从而成为化学预防和治疗的有效癌症靶点，还没有得到检验。本应用的目的是在生理环境下使用组织特异性SAG基因敲除小鼠模型，以研究SAG在原代培养物永生化和转化以及在肺和皮肤组织癌变中的作用。中心假说是，SAG在过度表达后，通过促进肿瘤抑制和凋亡诱导底物的降解，如IkB、Nf1、p27和Noxa，促进细胞生长和抑制细胞凋亡，导致NFkB和RAS通路的激活。另一方面，SAG在通过基因缺失或小分子抑制剂MLN4924处理失活时，会导致这些底物的积累，使NFkB和RAS途径失活，从而抑制增殖和致癌。为了阐明SAG在E1A/HRAS诱导的小鼠胚胎成纤维细胞永生化和转化中的作用，以及在KrasG12D诱导的肺癌和DMBA/TPA诱导的皮肤癌变中的作用。影响：我们的工作具有很高的创新性和显著的翻译价值，验证了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，从而为新发现的SCF E3连接酶小分子抑制剂MLN4924的未来开发提供了一些概念验证证据，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.