title: Inhibition of MDM2 homodimerization by XIAP IRES stabilizes MDM2, influencing cancer cell survival.
abstract: BACKGROUND
  It is known that the MDM2 protein is stabilized when it forms a heterodimer with its partner MDM4, but MDM2 protein stability in its homodimer form is not known. The MDM2 protein contains a C-terminal RING domain that not only functions as an E3 ligase to regulate ubiquitination of p53 and MDM2 itself, but also is characterized to be able to bind several specific cellular mRNAs to regulate gene expression. In this study, we evaluate whether the MDM2 protein stability is regulated by the binding of a specific small RNA (XIAP IRES mRNA).
  
  
  METHODS
  We performed chemical cross-linking and bimolecular fluorescence complementation (BiFC) assay to measure the human MDM2 protein stability in its homodimer form and the effect of XIAP IRES on MDM2 homodimerization and protein stabilization. Ubiquitination and pulse-chase assays were used to detect MDM2 self-ubiquitination and protein turn-over. Fluorescent titration and ITC were used to examine the binding between MDM2 RING protein and XIAP IRES. Western blot assay was used for determining protein expression. Clonogenic assay, WST and flow cytometry were used to test the effects of XIAP IRES, siXIAP and IR on cancer cell growth and apoptosis.
  
  
  RESULTS
  We found that self-association (homodimerization) of MDM2 occurs through the C-terminal RING domain of MDM2 and that the MDM2 protein becomes unstable when it is homodimerized. MDM2 homodimerization resulted in an increased function of the RING domain for MDM2 self-ubiquitination. Binding of XIAP IRES to the RING domain inhibited MDM2 homodimerization and self-ubiquitination, which resulted in stabilization of MDM2, as well as increased XIAP expression. Upregulation of XIAP and MDM2 that led to inhibition of p53 by the XIAP IRES resulted in cell growth and survival in both p53-normal and -deficient cancer cells.
  
  
  CONCLUSIONS
  Our study identified a new IRES RNA that interacts with MDM2 protein and regulates its stabilization, which suggested that targeting of MDM2 through disruption of MDM2 protein-RNA interaction might be a useful strategy for developing novel anti-cancer therapeutics.
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. tliu30@emory.edu.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. hzhan52@emory.edu.
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. xiongjing@126.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. sha.yi@emory.edu.
authors.forename: Sha
authors.initials: S
authors.lastname: Yi
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. mzhou@emory.edu.
authors.forename: Lubing
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Pediatrics and Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA, 30322, USA. lbgu@emory.edu.
authors.forename: Muxiang
authors.initials: M
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival / Q000235:genetics*; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics*; D055550:Protein Stability; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics*
chemical_list: D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2