NRF2 Is a Major Target of ARF in p53-Independent Tumor Suppression

Abstract

Although ARF can suppress tumor growth by activating p53 function, the mechanisms by which it suppresses tumor growth independently of p53 are not well understood. Here, we identified ARF as a key regulator of nuclear factor E2-related factor 2 (NRF2) through complex purification. ARF inhibits the ability of NRF2 to transcriptionally activate its target genes, including SLC7A11, a component of the cystine/glutamate antiporter that regulates reactive oxygen species (ROS)-induced ferroptosis. As a consequence, ARF expression sensitizes cells to ferroptosis in a p53-independent manner while ARF depletion induces NRF2 activation and promotes cancer cell survival in response to oxidative stress. Moreover, the ability of ARF to induce p53-independent tumor growth suppression in mouse xenograft models is significantly abrogated upon NRF2 overexpression. These results demonstrate that NRF2 is a major target of p53-independent tumor suppression by ARF and also suggest that the ARF-NRF2 interaction acts as a new checkpoint for oxidative stress responses.

Keywords: ARF; NRF2; ROS; ferroptosis; oxidative stress; p53; transcriptional regulation.

Figure 1. Identification of ARF as a component of the NRF2 protein complexes

A. Coomassie blue staining of affinity-purified protein complexes from nuclear extracts of a Flag-HA-NRF2/H1299 stable cell line (lane 2) and the parental H1299 cell line (lane 1). Specific NRF2-interacting protein bands were analyzed by mass spectrometry. B. Human 293 cells were co-transfected with expression vectors encoding NRF2-V5 and/or Flag-ARF, as indicated. Lysates were immunoprecipitated with FLAG/M2 beads, and the fractionated proteins were immunoblotted with antibody specific to the V5 (for NRF2, upper panel) or the Flag (for ARF, lower panel) epitope. C. Co-immunoprecipitation of ARF with NRF2 from H1299 cells. Whole cell extracts (lane 1) or immunoprecipitates generated with the NRF2 antibody (lane 3) or a control IgG (lane 2) were immunoblotted with anti-ARF (lower) or anti-NRF2 (upper) antibody. D. Co-immunoprecipitation of NRF2 with ARF from H1299 cells. Whole cell extracts (lane 1) and immunoprecipitates generated with the ARF antibody (lane 3) or a control IgG (lane 2) were immunoblotted with anti-ARF (upper) or anti-NRF2 (lower) antibody. E. Western blot analysis of an in vitro GST pull-down assays of highly purified FHNRF2 protein incubated with GST-ARF (lane 3) or GST alone (lane 2). F. Western blot analysis of an in vitro GST pull-down assays of highly purified FHNRF2 protein incubated with GST-ARF (1–64) (lane 3), GST-ARF (65–132) (lane 4) or GST alone (lane 2). G. H1299 cells were transfected with the SLC7A11-Luc reporter construct together with expression vectors encoding NRF2 and differing amounts of ARF. H. H1299 cells were transfected with the SLC7A11-Luc reporter construct together with expression vectors encoding NRF2 and either full-length HA-ARF, HAARF(1–64), or HA-ARF(65–132). See also Figure S1.

Figure 2. ARF regulates the transcriptional activity of NRF2 and promotes ferroptosis

A. U2OS cells were co-transfected with expression vectors encoding FH-NRF2 and either full-length ARF or ARF(Δ14), which lacks amino acids 1–14. Lysates were immunoprecipitated with FLAG-M2 beads, and the fractionated proteins were immunoblotted with HA-specific antibody (to detect FH-NRF2, upper panel) or ARF-specific antibody (lower panel). B. H1299 cells were transfected with the SLC7A11-Luc reporter construct, together with expression vectors encoding NRF2 and full-length ARF or ARFΔ14. C. Extracts of ARF-inducible SaoS2 cells treated with doxycycline were immunoblotted with antibodies specific for NRF2, SLC7A11, ARF and Actin. D. Extracts of SaoS2 cells treated with an ARF-specific RNAi (lane 2) or a control RNAi (lane 1) were immunoblotted with antibodies specific for NRF2, SLC7A11, ARF and Actin. E. Extracts of ARF-inducible H1299 cells treated with doxycycline as indicated were immunoblotted with antibodies specific for NRF2, SLC7A11, ARF and Actin. F. Representative phase-contrast images of ferroptotic cell death induced by ROS (TBH treatment) in ARF-inducible H1299 cells treated with or without doxycycline and/or Ferrostatin-1 (Ferr-1) as indicated. G. Quantification of ROS-induced cell death from Figure 2F (error bars, s.d. from three technical replicates). H. The percent cell death induced by ROS was determined for H1299 cells transfected with an empty expression vector or with expression vectors encoding ARF alone or ARF and NRF2 together (error bars, s.d. from three technical replicates). See also Figure S2.

Figure 3. ARF can induce both p53-dependent and p53-independent ferroptosis

A. ARF-inducible U2OS cells were treated with control RNAi (lane 1), p53 RNAi (lane 2), control RNAi + IPTG (lane 3), or p53 RNAi + IPTG (lane 4) as indicated, and cell extracts were immunoblotted with antibodies specific for NRF2, SLC7A11, p53, p21, ARF and Actin. B. Representative phase-contrast images of ARF-inducible U2OS cells treated with ROS and either IPTG (to induce ARF expression) and/or Ferrostatin-1 (Ferr-1), as indicated. C. Quantification of ROS-induced cell death of the cells shown in 3B (error bars, s.d from three technical replicates). D. Extracts of p53^fl/fl and ARF^fl/fl/p53^fl/fl MEFs treated with either the Ad-Cre or the control Ad-GFP virus were immunoblotted with antibodies specific for p53 and ARF. E. ARF is critical for p53-independent ferroptosis. Quantification of ferroptotic cell death in p53^fl/fl and ARF^fl/fl/p53^fl/fl MEFs treated with the Ad-Cre virus with or without Erastin as indicated (error bars, s.d. from three technical replicates). F. 293 cells were transfected with expression vectors encoding Flag-NRF2, CBP-HA, and/or ARF/ARFΔ14 as indicated. Lysates were immunoprecipitated with FLAG-M2 beads, and the fractionated proteins were immunoblotted with the acetylated specific antibody to acetylated NRF2 (upper), the Flag antibody (for Flag-NRF2; middle). ARF or ARFΔ14 in crude lysates were analyzed by western blotting with anti-ARF antibody (lower). G. ChIP-qPCR analysis of NRF2 binding on the endogenous SLC7A11 promoter. H1299 cells were transfected with expression vectors encoding FH-NRF2 and either full-length ARF or ARF(Δ14), which lacks amino acids 1–14. See also Figure S3.

Figure 4. The ARF-NRF2 interaction is critical for ARF-mediated tumor suppression independent of p53

A. Representative phase-contrast images of H₂O₂-treated ARF-inducible H1299 cells cultured in the presence or absence of doxycycline, and/or the presence of Ferrostatin-1 (Ferr-1) and/or Z-VAD-FMK, as indicated. B. Quantification of H₂O₂-induced cell death from the samples shown in Figure 4A (error bars, s.d from three technical replicates). C. Quantification of H₂O₂-induced cell death from H1299 cells co-transfected with an empty expression vector or with expression vectors encoding ARF alone or ARF and NRF2 together. D. Xenograft tumors from tet-on ARF-inducible H1299 cells transfected with an empty expression vector or an expression vector encoding NRF2. E. Tumor weight was determined from Figure 4D (error bars, SEM from six tumors). Independent experiments were repeated three times and representative data are shown. F. Western blot analysis of ARF, NRF2 and SLC7A11 protein levels in tet-on ARFinducible H1299 cells with or without NRF2 overexpression. See also Figure S4.