Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis
Hua Li, … , David G. Beer, Yi Sun
Hua Li, … , David G. Beer, Yi Sun
Published January 16, 2014
Citation Information: J Clin Invest. 2014;124(2):835-846. https://doi.org/10.1172/JCI70297.
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Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis

Abstract

Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine KrasG12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of KrasG12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.

Authors

Hua Li, Mingjia Tan, Lijun Jia, Dongping Wei, Yongchao Zhao, Guoan Chen, Jie Xu, Lili Zhao, Dafydd Thomas, David G. Beer, Yi Sun

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Figure 4

SAG siRNA knockdown inhibits growth and survival of human lung cancer cells via inactivation of NF-κB and mTOR.

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SAG siRNA knockdown inhibits growth and survival of human lung cancer ce...
(A–C). Growth of A549 human lung tumor cells upon SAG siRNA knockdown. Human lung cancer A549 cells were subjected to lentivirus-based siRNA silencing of SAG (Lt-SAG) along with scramble siRNA control (Lt-Con). Cells were tested by ATP-lite based proliferation assay (n = 3) (A); clonogenic survival assay (n = 3) (B); and soft agar assay (n = 3) (C). (D) Accumulation of tumor-suppressive proteins upon SAG knockdown: A549 cells were infected with Lt-SAG or Lt-Con, followed by immunoblotting using indicated Abs. (E) SAG knockdown blocks p65 nuclear translocation. Cells upon SAG knockdown were treated with or without TNF-α (100 ng/ml) for 1 hour, followed by nuclear fractionation and immunoblotting. Caspase-3 and PARP were used to demonstrate the purity of cytoplasm and nuclear fractions, respectively. (F). p65 DNA binding: nuclear fraction from indicated treatment is subjected to DNA-binding assay using TransAM NF-κB assay kit. (G). Luciferase reporter–based NF-κB activity assay. Lentiviral infected cells were transiently transfected with pNifty plasmid, along with renilla for transfection efficiency control. Cells were untreated or treated with TNF-α (10 ng/ml) and assayed for luciferase activity (n = 3). *P < 0.05; **P < 0.01.