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 Our strong published results and unpublished preliminary data, using mouse transgenic and knockout models and cell-based mechanistic studies, demonstrated that 1) SAG positively regulates angiogenesis: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.

Public Health Relevance

Targeted cancer therapy relies on thorough validation of cancer targets. We found that SAG E3 ubiquitin ligase is overexpressed in skin cancer and SAG inactivation selectively suppresses the growth of skin squamous cell carcinoma cells. In this study, we will mechanistically elucidate the role of Sag in skin carcinogenesis triggered by viral, chemical and physical carcinogens, using tissue-specific mouse KO models. We will also test the activity of MLN4924, a small molecule inhibitor of SAG E3 ligase against skin cancer. Thus, this study is of highly translational value by providing proof-of-concept evidence for future development of SAG inhibitor MLN4924 as a novel class of anti-cancer agent for the prevention and treatment of skin cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Johnson, Ronald L
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Zhou, Weihua; Xu, Jie; Zhao, Yongchao et al. (2014) SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization. Oncotarget 5:6746-55
Xiong, Xiufang; Zhao, Yongchao; Tang, Fei et al. (2014) Ribosomal protein S27-like is a physiological regulator of p53 that suppresses genomic instability and tumorigenesis. Elife 3:e02236
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Zhou, Weihua; Wei, Wenyi; Sun, Yi (2013) Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases. Cell Res 23:599-619
Zhao, Yongchao; Sun, Yi (2013) Cullin-RING Ligases as attractive anti-cancer targets. Curr Pharm Des 19:3215-25
Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi (2013) Role of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases in skin cancer. J Genet Genomics 40:97-106