Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis
Hua Li, Mingjia Tan, Lijun Jia, Dongping Wei, Yongchao Zhao, Guoan Chen, Jie Xu, Lili Zhao, Dafydd Thomas, David G. Beer, Yi Sun
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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Research Article Oncology

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 8

MLN4924 suppresses growth of lung cancer cells by targeting NF-κB and mTOR pathways and by accumulating tumor suppressor substrates.

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MLN4924 suppresses growth of lung cancer cells by targeting NF-κB and mT...
(A–C) A549 cells were treated with MLN4924 at the indicated concentrations. Growth suppression effect was tested by monolayer growth (n = 3) (A), clonogenic survival (n = 4) (B), and soft agar (C, n = 2). **P < 0.01. (D). Accumulation of tumor-suppressive proteins: A549 cells were treated with indicated concentrations of MLN4924 for 24 hours, followed by immunoblotting using indicated Abs. (E). MLN4924 treatment blocks p65 nuclear translocation. Cells after 24-hour treatment of MLN4924 (1 μM) were exposed to TNF-α (100 ng/ml) or control vehicle for 1 hour, followed by nuclear fractionation and immunoblotting. Caspase-3 and PARP were used to demonstrate the purity of cytoplasm and nuclear fractions. (F) Time-dependent induction of DEPTOR and inactivation of mTORC1: cells were treated with 1 μM MLN4924 as well as DMSO for indicated periods and subjected to immunoblotting.