Role of SAG/RBX2 E3 Ubiquitin Ligase in Skin Carcinogenesis

描述(由申请者提供)：靶向癌症治疗依赖于对癌症靶点的彻底验证。我们的长期目标是发现一类新的抗癌药物，选择性地针对一种类型的E3泛素连接酶，该酶被证明在人类癌症中被激活。为此，我们重点研究了凋亡敏感基因SAG，也被称为RBX2/ROC2，它是SCF(Skp1-cullin-F-box Proteins)E3泛素连接酶的一个环成分，是其活性所必需的。我们已发表的结果和未发表的初步数据，通过使用小鼠转基因和基因敲除模型以及基于细胞的机制研究，证明了1)SAG正向调节血管生成：在皮肤肿瘤中，SAG转基因表达促进血管生成，在胚胎发育过程中，在ES细胞来源的畸胎瘤和B16黑色素瘤中，SAG基因敲除抑制血管生成；2)SAG E3促进a)神经纤维蛋白1(NF1)的降解以激活RAS/MAPK，b)IKB激活NFkB，c)DEPTOR激活mTOR，从而促进增殖和生存；3)siRNA或小分子SAG E3抑制剂MLN4924使SAG失活，选择性地抑制人鳞状细胞癌(SCC)细胞的生长；4)SAG KO抑制角质形成细胞的分化，最后5)SAG在人皮肤SCC的三个发育阶段(光化性角化病、原位鳞癌和侵袭性SCC)中均过表达。然而，SAG在病毒(HPV16灭活P53和Rb)、化学物质(DMBA-TPA激活RAS-AP1)或物理致癌物(UV)诱导的皮肤癌发生中是否发挥重要作用，从而成为抗皮肤癌治疗的有效靶点，尚未得到系统研究。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，从机制上研究SAG在各种致癌物引发的皮肤癌中的作用。中心假说是SAG通过靶向降解肿瘤抑制底物NF1、I？B和DEPTOR，分别激活RAS、NF？B和mTOR通路，从而促进血管生成、增殖和肿瘤发生。通过基因缺失或药物抑制剂MLN4924使SAG失活，会导致这些底物的积聚，使RAS、NF？B、mTOR途径失活，从而抑制皮肤癌变。通过确定SAG缺失对1)病毒癌基因诱导的皮肤血管生成和致癌的影响，2)DMBA/TPA或UV诱导的皮肤癌变，以及3)SAG的作用机制，提出了三个特定的目标来验证我们的假说。影响：我们的工作使用皮肤特异的KO小鼠模型，概括了人类皮肤SCC的发展过程，从机制上阐明SAG E3连接酶在皮肤癌的发生中是必不可少的，从而成为治疗皮肤癌的有吸引力的药物靶点。我们的工作具有很高的创新性，具有重大的翻译价值，为MLN4924作为一种用于皮肤癌预防和治疗的新型抗癌药物的未来开发提供了概念验证证据。

DESCRIPTION (provided by applicant): Targeted cancer therapy relies on thorough validation of cancer targets. Our long-range goal is to discover a novel class of anticancer drugs that selectively target one type of E3 ubiquitin ligase, shown to be activated in human cancers. To this end, we have focused on SAG (Sensitive to Apoptosis Gene), also known as RBX2/ROC2, a RING component of SCF (Skp1-Cullin-F-box proteins) E3 ubiquitin ligases, required for its activity. Our strong published results and unpublished preliminary data, using mouse transgenic and knockout models and cell-based mechanistic studies, demonstrated that 1) SAG positively regulates angiogenesis: angiogenesis is enhanced by SAG transgenic expression in skin tumors, and inhibited by Sag knockout during embryonic development, in teratomas derived from ES cells, and in B16 melanoma tumorigenesis; 2) SAG E3 promotes the degradation of a) neurofibromin 1 (NF1) to activate the RAS/MAPK, b) IkB to activate NFkB, c) DEPTOR to activate mTOR, leading to enhanced proliferation and survival; 3) SAG inactivation by siRNA knockdown or by small molecule SAG E3 inhibitor MLN4924 selectively suppresses the growth of human squamous cell carcinoma (SCC) cells; 4) Sag KO inhibits keratinocyte differentiation, and finally 5) SAG is overexpressed in all three developmental stages of human skin SCC (namely, actinic keratosis, SCC in situ, and invasive SCC). However, whether Sag plays an essential role in skin carcinogenesis induced by viral (HPV16 to inactivate p53 and RB), chemical (DMBA-TPA to activate Ras-AP1), or physical (UV) carcinogens, thus serving as a valid target for anti-skin cancer therapy, has not been systematically examined. The objectives of this application are to use tissue specific Sag knockout mouse models under physiological settings to investigate mechanistically the role of Sag in skin carcinogenesis triggered by various carcinogens. The central hypothesis is that SAG promotes angiogenesis, proliferation and tumorigenesis via targeted degradation of tumor suppressive substrates such as NF1, I?B and DEPTOR, leading to activation of the RAS, NF?B and mTOR pathways, respectively. Inactivation of SAG by genetic deletion or pharmaceutical inhibitor MLN4924 would cause the accumulation of these substrates to inactivate the RAS, NF?B, mTOR pathways leading to suppression of skin carcinogenesis. Three specific aims are proposed to test our hypothesis by determining the effect of Sag deletion on 1) skin angiogenesis and carcinogenesis, induced by viral oncogenes; 2) skin carcinogenesis, induced by DMBA/TPA or UV; and 3) mechanisms of Sag action. IMPACT: Our work uses skin-specific KO mouse models that recapitulate the development of human skin SCC to elucidate mechanistically that SAG E3 ligase is essential for skin carcinogenesis, thus serving as an attractive drug target for skin cancer. Our work is highly innovative and of significant impact with translational value by providing proof-of-concept evidence for future development of MLN4924 as a novel class of anti-cancer agent for the prevention and treatment of skin cancer.