Role of SAG/RBX2 E3 Ubiquitin Ligase in Skin Carcinogenesis

描述（由申请人提供）：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种新的抗癌药物，选择性地靶向一种E3泛素连接酶，这种酶在人类癌症中被激活。为此，我们关注了SAG (Sensitive To Apoptosis Gene)，也被称为RBX2/ROC2，它是SCF （Skp1-Cullin-F-box蛋白）E3泛素连接酶的一个环组分，是其活性所必需的。通过小鼠转基因和敲除模型以及基于细胞的机制研究，我们强有力的已发表的结果和未发表的初步数据表明：1)SAG正调控血管生成：在皮肤肿瘤中，SAG转基因表达可增强血管生成，而在胚胎发育、胚胎干细胞衍生的畸胎瘤和B16黑色素瘤发生过程中，SAG敲除可抑制血管生成；2) SAG E3促进a)神经纤维蛋白1 （NF1）降解激活RAS/MAPK， b) IkB激活NFkB， c) DEPTOR激活mTOR，从而增强细胞增殖和存活；3) siRNA敲低或小分子SAG E3抑制剂MLN4924使SAG失活可选择性抑制人鳞状细胞癌（SCC）细胞生长；4) Sag KO抑制角质细胞分化，最后5)Sag在人类皮肤鳞状细胞癌的所有三个发育阶段（即光化性角化病、原位鳞状细胞癌和侵袭性鳞状细胞癌）均过表达。然而，Sag是否在病毒（HPV16灭活p53和RB）、化学（dba - tpa激活Ras-AP1）或物理（UV）致癌物诱导的皮肤癌变中发挥重要作用，从而作为抗皮肤癌治疗的有效靶点，尚未得到系统的研究。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在各种致癌物引发的皮肤癌变中的机制作用。核心假设是，SAG通过靶向降解肿瘤抑制底物（如NF1、i - κ b和DEPTOR）促进血管生成、增殖和肿瘤发生，从而分别激活RAS、nf - κ b和mTOR通路。通过基因缺失或药物抑制剂MLN4924使SAG失活，会引起这些底物的积累，使RAS、NFκB、mTOR通路失活，从而抑制皮肤癌的发生。为了验证我们的假设，我们提出了三个具体的目标，即确定Sag缺失对1)由病毒致癌基因诱导的皮肤血管生成和致癌的影响；2) DMBA/TPA或UV诱发的皮肤癌变；3)凹陷作用机理。影响：我们的工作使用皮肤特异性KO小鼠模型，再现了人类皮肤SCC的发展，以阐明SAG E3连接酶对皮肤癌变至关重要的机制，因此可以作为一个有吸引力的皮肤癌药物靶点。我们的工作具有高度的创新性，为MLN4924作为预防和治疗皮肤癌的新型抗癌药物的未来发展提供了概念验证证据，具有重要的转化价值。

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 Our strong published results and unpublished preliminary data, using mouse transgenic and knockout models and cell-based mechanistic studies, demonstrated that 1) SAG positively regulates angiogenesis: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, IκB and DEPTOR, leading to activation of the RAS, NFκB 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, NFκB, 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.