title: Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target.
abstract: OBJECTIVE
  Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity.
  
  
  METHODS
  SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins.
  
  
  RESULTS
  SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life.
  
  
  CONCLUSIONS
  The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target.
authors.affiliation: Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, USA.
authors.forename: Lijun
authors.initials: L
authors.lastname: Jia
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Xinbao
authors.initials: X
authors.lastname: Hao
authors.forename: Min
authors.initials: M
authors.lastname: Zheng
authors.forename: Hongbin
authors.initials: H
authors.lastname: He
authors.forename: Xiufang
authors.initials: X
authors.lastname: Xiong
authors.forename: Liang
authors.initials: L
authors.lastname: Xu
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016503:Drug Delivery Systems; D020868:Gene Silencing; D006801:Humans; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011838:Radiation-Sensitizing Agents; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation
chemical_list: C498702:PMAIP1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases; D053148:Caspase 3