Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Abstract

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2-5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-Ras(G12D), B-Raf(V619E) and Myc(ERT2) each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-Ras(G12D) and B-Raf(V619E), and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-Ras(G12D)-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

Figure 1. Physiological expression of oncogenes lowers ROS

NIH3T3s and MEFs were transduced with retroviral vectors and evaluated 6 days later: control vector (pBabe), pBabe-H-Ras^G12V (p-Babe-H-Ras), or pBabe-K-Ras^G12D (p-Babe-K-Ras). Alternatively, LSL-K-Ras^G12D MEFs were infected with Ad-mock (K-Ras^LSL/+) or Ad-cre (K-Ras^G12D/+) and evaluated 4 days later. Wild-type MEFs were infected with Ad-mock (WT Ad-Mock) or Ad-cre (WT Ad-Cre) and used as controls. a, (Left) Western blot of total and GTP-bound Ras in MEFs expressing endogenous and ectopic Ras, with Rac used as a loading control. (Right) ROS levels following expression of oncogenic Ras, as determined by 2′,7′-dichlorofluorescein diacetate (DCF) staining. b, 8-oxo-dGuo levels following ectopic and endogenous expression of K-Ras^G12D. c-f, Determination of the GSH/GSSG ratios and total cellular glutathione in cells overexpressing ectopic K-Ras^G12D (c,d), or expressing endogenous K-Ras^G12D (e,f). g, ROS levels following activation of Myc^ERT2. R26^MER/MER MEFs were treated with DMSO or 100nM 4-OHT and assayed after 24 hours. Data are representative of 3 or more independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and error bars represent ± SEM here and for all figures.

Figure 2. Physiological expression of oncogenes activates the Nrf2 antioxidant program

a, Western blot demonstrates a 60% increase in Nrf2 protein following expression of endogenous K-Ras^G12D. Antibody specificity was confirmed using Nrf2^-/- MEFs. b, Nrf2 ChIP followed by q-PCR for the Hmox1 and Nqo1 promoters. Control non-specific primers amplified regions of DNA located 50Kb from the Hmox1 and Nqo1 promoters. c, Expression of Nrf2 and Nrf2 target genes Nqo1, Hmox1, Gclm, Gclc and Ggt1 upon K-Ras^G12D expression in Nrf2^+/+ and Nrf2^-/- MEFs. Nrf2 mRNA is relatively unstable but still detectable at low levels in Nrf2^-/- MEFs. d-e, Determination of the GSH/GSSG ratio (d) and total glutathione (e) upon K-Ras^G12D expression in Nrf2^-/- MEFs. f, ROS levels following Nrf2 depletion with siRNA. LSL-K-Ras^G12D MEFs were transfected with non-targeting (NT) or Nrf2 siRNA, infected with Ad-mock or Ad-cre and assayed after 48 hours for DCF oxidation. g, Western blot of Nrf2 protein levels following induction of Myc^ERT2 by 4-OHT. Densitometry shows a 2.3-fold increase. h, Analysis of Nrf2 antioxidant program gene expression following activation of Myc^ERT2. R26^MER/MER MEFs were treated with DMSO or 100nM 4-OHT for 24 hours and assayed for antioxidant gene expression. Data is representative of 3 independent experiments.

Figure 3. Activation of Nrf2 by K-Ras^G12D occurs via the Raf-MEK-ERK-Jun pathway

a, ROS levels following treatment of K-Ras^G12D/+ MEFs with AZD6244. LSL-K-Ras^G12D MEFs were treated with DMSO or 0.1uM AZD6244, infected with Ad-mock (K-Ras^LSL/+) or Ad-cre (K-Ras^G12D/+) and assayed after 72 hours. b, Analysis of antioxidant gene expression following treatment of K-Ras^LSL/+ and K-Ras^G12D/+ MEFs with AZD6244 for 24 hours. c, Control of Nrf2 transcription by AP-1 family members. K-Ras^LSL/+ and K-Ras^G12D/+ MEFs were transfected with siRNA and assayed for Nrf2 expression after 48 hours. d, Western blot of Jun and actin protein levels in LSL-K-Ras^G12D and LSL-B-Raf^V619E MEFs. K-Ras^LSL/+ and K-Ras^G12D/+ MEFs were treated with DMSO or 0.1uM AZD6244 for 24 hours. e, ROS levels following Jun depletion with siRNA. LSL-K-Ras^G12D MEFs were transfected with non-targeting (NT) or Jun siRNA, infected with Ad-mock or Ad-cre and assayed after 48 hours for DCF oxidation. Data are representative of 3 independent experiments.

Figure 4. Evidence for Nrf2 antioxidant program in pancreatic cancer

a, Immunohistochemical detection of Nqo1 (brown staining) and 8-oxo-dGuo (purple staining) in mouse PanIN and PDA (similar patterns observed for 11/11 of cases examined) in comparison to morphologically normal ducts. PanIN (arrows), PDA (black arrowheads), normal ducts (white arrowheads) here and for all figures. Scale bar = 56 μm. b, Immunohistochemical detection of Nqo1 and 8-oxo-dGuo in Nrf2^-/- PanIN compared to Nrf2^+/+ PanIN (similar patterns observed for 5/5 of each genotype examined, PanIN outlined by white dashes). Scale bar = 56 μm. c, Nrf2^-/- and Nrf2^+/+ PanIN-1a incidence. Whole pancreata were sectioned at 100-micron intervals and total numbers of PanIN-1a were counted. d, Proliferation of PanIN-1a cells in Nrf2^-/- and Nrf2^+/+ mice, as determined by Ki-67 immunostaining.