Molecular and Cellular Pathobiology Proteomic Analysis of Ubiquitin Ligase KEAP 1 Reveals Associated Proteins That Inhibit NRF 2 Ubiquitination

Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2, and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acidmotif, whichmatches the KEAP1 interactionmotif found in NRF2. LikeWTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an "ETGE"motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1-interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA overexpression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 overexpression in the presence of thesemutants further promotes NRF2 activation. Collectively, our findings further support the competitionmodel of NRF2 activation and suggest that "ETGE"-containing proteins such as DPP3 contribute to NRF2 activity in cancer. Cancer Res; 73(7); 2199–210. 2013 AACR. Introduction Constitutive activation of the NF-E2–related factor 2 (NRF2) cap-n-collar transcription factor is emerging as a prominent molecular feature ofmany tumors.When active, NRF2 controls the expression of 200 genes that collectively function to maintain a healthy intracellular reduction–oxidation (redox) balance, clear electrophilic xenobiotics, and degrade damaged and misfolded proteins (1, 2). The leading hypothesis posits that whereas short-term NRF2 activation antagonizes oncogenesis by curtailing oxidative damage, constitutive activation promotes the survival of metabolically stressed cancer cells, as well as cancer cells under chemotherapeutic insult. Indeed, depletion of NRF2 from cancer-derived cell lines results in apoptosis and increased sensitivity to chemotherapeutic agents (3). In human non–small cell lung cancer, tumors showing high levels of NRF2 protein are associated with a poor outcome and increased resistance to therapy (4–6). At basal state, NRF2 protein level and activity is maintained at low levels through ubiquitin-dependent proteosomal degradation (7–9). The mechanics of this ubiquitination, which is conceptualized in the 'hinge-and-latch' model, involves a homodimeric E3 ubiquitin ligase complex comprising the KEAP1 substrate recognition module and a cullin-3 scaffold (refs. 10 and 11; Fig. 1A). An amino-terminal DLG and ETGE motif within NRF2 independently binds 2 KEAP1 monomers within the complex, yielding a 2:1 stoichiometry of KEAP1: NRF2. The intermolecular protein dynamics governing ubiquitination of NRF2 relies on the differential affinities between the ETGE andDLGmotifs for KEAP1; the ETGEmotif binds KEAP1 with approximately 100-fold greater affinity than the DLG (10). In response to oxidative stress, modification of reactive cysteines within KEAP1 induces a conformational change within the homodimer. This architectural restructuring releases the low-affinity DLG motif from KEAP1, thus repositioning NRF2 in a conformation unfavorable for ubiquitination (10–13). Recent cancer genomic studies reported somatic mutation of NRF2 or KEAP1 in 34% of squamous cell lung carcinoma and Authors' Affiliations: Department of Cell Biology and Physiology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine; Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill; Department of Internal Medicine and Otolaryngology, Division of Medical Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill; and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). D. Goldfarb and K.M. Mulvaney contributed equally to this work. Corresponding Author: Michael B. Major, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Box #7295, Chapel Hill, NC 27599. Phone: 919-966-9258; Fax: 919-966-8212; E-mail: benmajor@med.unc.edu doi: 10.1158/0008-5472.CAN-12-440