title: Depletion of SAG/RBX2 E3 ubiquitin ligase suppresses prostate tumorigenesis via inactivation of the PI3K/AKT/mTOR axis.
abstract: SAG (Sensitive to Apoptosis Gene), also known as RBX2, ROC2 or RNF7, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our recent study showed that SAG/RBX2 co-operated with Kras to promote lung tumorigenesis, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. However, it is totally unknown whether and how Sag would play in prostate tumorigenesis, triggered by Pten loss.
  
  
  
  Sag and Pten double conditional knockout mice were generated and prostate specific deletion of Sag and Pten was achieved by PB4-Cre, and their effect on prostate tumorigenesis was evaluated by H&E staining. The methods of immunohistochemistry (IHC) staining and Western blotting were utilized to examine expression of various proteins in prostate cancer tissues or cell lines. The effect of SAG knockdown in proliferation, survival and migration was evaluated in two prostate cancer cell lines. The poly-ubiquitylation of PHLPP1 and DEPTOR was evaluated by both in vivo and in vitro ubiquitylation assays.
  
  
  
  SAG is overexpressed progressively from early-to-late stage of human prostate cancer with the highest expression seen in metastatic lesion. Sag deletion inhibits prostate tumorigenesis triggered by Pten loss in a mouse model as a result of suppressed proliferation. SAG knockdown in human prostate cancer cells inhibits a) proliferation in monolayer and soft agar, b) clonogenic survival, and c) migration. SAG is an E3 ligase that promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis, whereas SAG knockdown caused their accumulation. Importantly, growth suppression triggered by SAG knockdown was partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR, suggesting their causal role. Accumulation of Phlpp1 and Deptor with corresponding inactivation of Akt/mTOR was also detected in Sag-null prostate cancer tissues.
  
  
  
  Sag is an oncogenic cooperator of Pten-loss for prostate tumorigenesis. Targeting SAG E3 ligase may, therefore, have therapeutic value for the treatment of prostate cancer associated with Pten loss.
authors.affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, 4424B MS-1, 1301 Catherine Street, Ann Arbor, 48109, MI, USA.
authors.forename: Mingjia
authors.initials: M
authors.lastname: Tan
authors.affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, 4424B MS-1, 1301 Catherine Street, Ann Arbor, 48109, MI, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Pathology, University of Michigan, 4424B MS-1, 1301 Catherine Street, Ann Arbor, 48109, MI, USA.
authors.forename: Javed
authors.initials: J
authors.lastname: Siddiqui
authors.affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, 4424B MS-1, 1301 Catherine Street, Ann Arbor, 48109, MI, USA.|Department of Radiation Oncology, University of San Francisco, San Francisco, CA, USA.
authors.forename: Felix
authors.initials: F
authors.lastname: Feng
authors.affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, 4424B MS-1, 1301 Catherine Street, Ann Arbor, 48109, MI, USA. sunyi@umich.edu.|Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People's Republic of China. sunyi@umich.edu.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, People's Republic of China. sunyi@umich.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D002352:Carrier Proteins; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C573100:SAG protein, mouse; C546843:mTOR protein, mouse; C487757:Pten protein, mouse
keywords: DEPTOR; PHLPP1; Prostate tumorigenesis; Pten; SAG KO; SAG-SCF E3; Ubiquitin ligase