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Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer1. 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 and its repressor protein Keap12-5. 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, suggesting that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic6. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 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 novel mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find 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. Oncogenedirected increased expression of Nrf2 is a novel 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.
To examine the role of ROS in cellular transformation and tumorigenesis, we utilized an endogenous and conditional oncogenic LSL-K-Ras G12D allele 7 . K-Ras G12D/+ and K-Ras LSL/+ MEFs were compared to MEFs and NIH3T3 fibroblasts (NIH3T3s) transduced with K-Ras4B G12D and H-Ras V12 (Fig. 1a), since ectopic Ras introduction was previously reported to increase ROS production 8 . In contrast to ectopic overexpression of oncogenic Ras, K-Ras G12D/+ MEFs demonstrated lower levels of hydrogen peroxide, superoxide, and mitochondrial ROS compared to K-Ras LSL/+ MEFs (Fig. 1a, Supplementary Fig. 2a,b). Accordingly, the level of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo), one of the major products of DNA oxidation, was decreased in MEFs expressing endogenous K-Ras G12D/+ , but increased by ectopic oncogenic Ras (Fig. 1b). The levels of ROS and 8-oxo-dGuo were also lower in p53 -/-; K-Ras G12D/+ MEFs compared to p53 -/-; K-Ras LSL/+ MEFs ( Supplementary Fig. 2c,d), supporting that these differences are not due to differential activation of a senescence program and demonstrating that increased ROS production is not required for full transformation 7,9 . Because alterations of ROS can affect the intracellular redox state 10 , the ratio of reduced/ oxidized glutathione (GSH/GSSG) was examined in cells expressing ectopic and endogenous K-Ras G12D . While both elevated the level of total glutathione (Fig. 1d,f and ref. 11), only the endogenous expression of K-Ras G12D elevated the GSH/GSSG ratio to promote a more reduced intracellular environment (Fig. 1c,e). The production of ROS by ectopic oncogenic Ras is chiefly regulated by NADPH-oxidase (Nox) 12 . Only the ectopic overexpression of oncogenic Ras increased Nox activity and mRNA, providing a mechanistic explanation for these differing results ( Supplementary Fig. 3). Similar to oncogenic Ras, ectopic expression of Myc has also been shown to increase the production of ROS in NIH3T3 fibroblasts 13 and accordingly we sought to determine whether nearphysiological expression of this oncogene had a similar effect. Treatment of R26 MER/MER MEFs, which express a homozygous Myc ERT2 allele under the control of the Rosa26 promoter 14 , with 4-OHT resulted in a substantial reduction of ROS (Fig. 1g). However, this was not a characteristic feature of all oncogenic stimuli as expression of activated Notch1 and β-catenin did not lower ROS ( Supplementary Fig. 4). Therefore, endogenous expression of oncogenic K-Ras or Myc ERT2 lowers the level of cellular ROS, in contrast to the ectopic overexpression of these oncogenes.
As the intracellular redox state is chiefly regulated by Nrf2, we examined Nrf2 protein levels and activity in K-Ras LSL/+ and K-Ras G12D/+ MEFs by western blot, reporter assay and chromatin immunoprecipitation analysis of the Nrf2 target genes Hmox1 and Nqo1. Expression of K-Ras G12D resulted in an approximately two-fold increase in Nrf2 protein and binding activity compared to control MEFs (Fig. 2a,b, Supplementary Fig. 5a), which was not attributable to altered regulation by Keap1 2 (Supplementary Fig. 6). K-Ras G12D/+ MEFs demonstrated increased Nrf2 mRNA and increased expression of Hmox1, Nqo1, Gclc, Gclm and Ggt1 mRNA and protein (Fig. 2c, Supplementary Fig. 5b). Increased expression of Nrf2 and its target genes was also observed following K-Ras G12D expression in p53 -/-MEFs, and following ectopic expression of K-Ras G12D and H-Ras V12 in primary MEFs, but not following expression of activated Notch1 or β-catenin (Supplementary Figs. 5c-g). However, expression of K-Ras G12D in Nrf2-deficient MEFs failed to elevate total glutathione and resulted in a more oxidized intracellular environment (Fig. 2d,e). Neither the cell culture conditions employed to express K-Ras G12D nor the gene dosage of wild-type K-Ras affected the expression of Nrf2 target genes ( Supplementary Fig. 7a,b). Additionally, ROS metabolism in wild-type MEFs was sensitive to acute changes in the levels of Keap1 and Nrf2, further supporting a causal relationship between Nrf2 and ROS (Supplementary Fig. 7c-e). Furthermore, acute knockdown of Nrf2 attenuated the reduction in ROS by K-Ras G12D (Fig. 2f), and the effects of Nrf2 depletion on ROS were dosage-dependent, supporting the importance of the level of Nrf2 mRNA for ROS control ( Supplementary Fig.  7f,g). Similar to K-Ras G12D , activation of c-Myc ERT2 (with 4-OHT) promoted an increase in the mRNA and protein levels of Nrf2 and its target genes (Fig. 2g,h, Supplementary Fig.  7h). Furthermore, ChIP-seq data from the ENCODE consortium demonstrated direct binding of Myc to the Nrf2 locus ( Supplementary Fig. 8a) 15 . Therefore, the K-Ras and Myc oncogenes can constitutively increase the transcription of Nrf2 to elevate the basal activity of the antioxidant and cellular detoxification program.
To investigate the mechanism of Nrf2 activation by K-Ras G12D , the roles of the Raf/MEK/ERK and p38alpha MAPK pathways were investigated. First, cells were treated with a potent and specific inhibitor of MEK, AZD6244 (ARRY-142886) (Supplementary Fig. 9a-c), which restored the ROS level of K-Ras G12D/+ cells nearly to the level of K-Ras LSL/+ cells (Fig. 3a). Additionally, AZD6244 treatment resulted in decreased induction of Nrf2 and its target genes (Fig. 3b). Furthermore, endogenous expression of B-Raf V619E (corresponding to human B-Raf V600E ) 16 resulted in increased phospho-ERK levels, a decrease in ROS, and an increase in Nrf2 mRNA and antioxidant gene expression (Supplementary Fig. 9d-f). As previously reported 17 , we found that p38alpha MAPK kinase did not activate Nrf2 ( Supplementary Fig. 9g-i). To determine the mechanism of increased Nrf2 expression, transcription factors downstream of MAPK signaling were examined. Accordingly, knockdown of Jun, Fra1, and Myc, but not JunD or Elk1, decreased the Nrf2 mRNA in K-Ras G12D/+ cells, with almost complete rescue achieved with Jun (Fig. 3c). siRNA efficiency was confirmed by real-time PCR and western blot ( Supplementary Fig.  10a,b). Importantly, K-Ras G12D/+ MEFs and B-Raf V619E MEFs demonstrated elevated Jun protein levels compared to control MEFs, and the elevated Jun level in K-Ras G12D/+ MEFs was rescued by treatment with AZD6244 (Fig. 3d). Furthermore, depletion of Jun with siRNA prevented the decrease in ROS following expression of K-Ras G12D (Fig. 3e,  Supplementary Fig. 10c). Our results reinforce a prior finding that antioxidant response elements that closely resemble AP-1 sites help regulate the Nrf2 promoter 18 . Finally, ChIPseq revealed that the transcriptional start site of the Nrf2 locus is a direct binding target of Jun ( Supplementary Fig. 8b) 15 . Thus, K-Ras G12D and B-Raf V619E stimulate transcription of Nrf2 via Jun and Myc.
We next examined whether these oncogenes promote activation of the Nrf2 antioxidant and cellular detoxification program in vivo. Elevated protein expression of the Nrf2 target gene Nqo1 and decreased immunoreactivity for 8-oxo-dGuo were evident in K-Ras mutant murine 19 and human preinvasive pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDA), and in murine lung adenomas expressing B-Raf V619E (Fig.  4a, Supplementary Figs. 11a, 12, 13, 14a). PanIN also demonstrated reduced immunoreactivity for the lipid peroxidation adduct malondialdehyde (MDA) ( Supplementary Fig. 11b). Furthermore, K-Ras G12D/+ pancreatic epithelial cells demonstrated activation of Nrf2 and decreased ROS compared to K-Ras LSL/+ , while treatment of human PDA tumor lines with K-Ras siRNA resulted in decreased Nrf2 and Nqo1 mRNA and an increase in ROS ( Supplementary Fig. 15). The activation of Nrf2 in human pancreatic cancer could not be explained by somatic mutations in NRF2 or KEAP1; we sequenced over 100 samples of human PDA and identified only one case with a concomitant KEAP1 and K-RAS mutation (Supplemental material and Supplementary Fig.  14b). Importantly, Nrf2-deficient murine PanIN were negative for Nqo1 and demonstrated similar levels of 8-oxo-dGuo and MDA in PanIN compared to neighboring morphologically normal ductal cells, supporting a role for Nrf2 in ROS detoxification in vivo (Fig. 4b, Supplementary Fig. 11b,c). Nrf2-deficient salivary gland did not demonstrate elevated 8oxo-dGuo immunoreactivity, suggesting that K-Ras G12D -expressing cells are more reliant on Nrf2 for ROS detoxification (Supplementary Fig. 16a). Therefore, an antioxidant program is operant during tumorigenesis, consistent with our findings that link oncogenic K-Ras and B-Raf expression with the activation of Nrf2.
To investigate whether activation of Nrf2 promotes K-Ras G12D -initiated carcinomas, the effects of Nrf2 ablation were examined in established mouse models of pancreatic and lung cancer. Nrf2-deficient pancreata were noted to contain fewer PanIN (Fig. 4c). While the proliferation of the salivary gland was unchanged ( Supplementary Fig. 16b), PanIN from Nrf2-deficient mice were significantly less proliferative and demonstrated an increased content of cells exhibiting senescence-associated β-galactosidase activity (Fig. 4d,  Supplementary Fig. 17a). No significant differences were observed in PanIN cell apoptosis or DNA damage ( Supplementary Fig. 17b,c). Senescence was also observed in K-Ras G12D/+ MEFs following acute depletion of Nrf2 with siRNA and was dependent on expression of p53 ( Supplementary Fig. 17d). The Nrf2 dependent proliferation defect noted in PanIN was also observed in MEFs, and both could be rescued by the antioxidant N-acetyl cysteine ( Supplementary Fig. 18a,b). Furthermore, treatment of Nrf2 +/+ PanIN mice with a glutathione synthesis inhibitor resulted in a significant decrease in the proliferation of PanIN but not adjacent acinar cells ( Supplementary Fig. 18c-e). Additionally, the role of Nrf2 in lung cancer development was investigated. Nrf2 deficiency resulted in a significant reduction in disease burden and proliferation, and an increase in median survival ( Supplementary Fig. 19). While we cannot exclude the potential involvement of non-cell autonomous effects arising from constitutive Nrf2 deficiency, the major impact appeared to be restricted to tumor initiation and proliferation of nascent preneoplastic cells. These results demonstrate that Nrf2 promotes K-Ras G12D -initiated pancreatic and lung tumorigenesis and proliferation.
ROS can stimulate tumorigenesis through the oxidation of DNA and subsequent mutation of genes that promote carcinogenesis. In contrast, we found that activating an ROSdetoxification program contributed to tumorigenesis, although our study does not exclude a role for other Nrf2 targets such as drug-metabolizing enzymes and efflux pumps, heat shock proteins, 26S proteasome subunits, growth factors and receptors 6 . Future studies will be necessary to determine the role of these Nrf2 functions in tumors with activated Nrf2. Increased expression of Nrf2 target genes and increased stability of Nrf2 caused by somatic mutations in Nrf2 and Keap1 are well documented in human cancer, providing one mechanism for enhanced Nrf2 activity during tumorigenesis. Our work describes oncogenic signaling as an alternative mechanism to activate Nrf2 transcription during tumorigenesis ( Supplementary Fig. 1), and suggests that modulation of the redox state is uniformly important in cancer and may therefore represent a therapeutic opportunity. Thus, constitutively elevated Nrf2 activity in cancer cells occurs through two distinct mechanisms: diminished Nrf2 turnover and augmented Nrf2 mRNA levels.

ChIP-seq (ENCODE)
These data were generated and analyzed by the labs of Michael Snyder, Mark Gerstein and Sherman Weissman at Yale University; Peggy Farnham at UC Davis; and Kevin Struhl at Harvard using the K562 cell line. The following antibodies were used: c-Jun (sc-1694, Santa Cruz), c-Myc (sc-764, Santa Cruz). The data can be accessed at http://genome.ucsc.edu/ ENCODE/

Human pancreatic cancer cell culture
MiaPaCa2 and Hs766T cells were obtained from CRUK Clare Hall Laboratories. Both lines were grown in DMEM + 10% FBS. siRNA transfections were performed with DharmaFECT 1.

Immunohistochemistry
Human tissue was donated for research purposes by patients undergoing pancreatic surgery at a tertiary hepatopancreaticobiliary referral center (Addenbrookes Teaching Hospitals NHS Trust, Cambridge, UK). Ethical approval was granted by the local research and ethics committee (LREC Number: 08/H0306/32). Paraffin embedded mouse and human specimens were deparaffinized and rehydrated, followed by antigen retrieval in boiling citrate buffer. The following primary antibodies were used: Ki-67 (SP-6, Neomarkers), Nqo1 (Abcam, Sigma Aldrich), 8-oxo-dGuo ([N45.1], Abcam), γ -H2AX (Millipore), Cleaved caspase 3 (Cell Signaling), anti-Malondialdehyde (Abcam). HRP-based detection reagents were used for all IHC except 8-oxo-dGuo, for which AP-based reagents were used. 8-oxo-dGuo specificity was verified by treating sections for 1 hour with 1U/ml DNase at 37°C. For quantification of 8-oxo-dGuo, mean signal intensity was calculated using Adobe Photoshop CS. Immunohistochemistry images were converted to grayscale images, inverted, and mean pixel 8-oxo-dGuo intensity analyzed. For murine Nqo1 IHC, the Sigma Aldrich anti-Nqo1 antibody was used, and the staining pattern was confirmed to be reproducible with the Abcam antibody (not shown). The Abcam Nqo1 antibody was used for human tissue.

Luciferase assay
LSL-K-Ras G12D and Nrf2 -/-; K-Ras G12D MEFs were plated in 12 well dishes at a density of 5 × 10 4 cells/well and infected with a Cignal Lenti ARE Reporter (Qiagen) at an MOI of 10. After 6 hours the cells were split 1:2 and infected the next day with Ad-mock and Ad-cre. After 4 days the cells were plated at 70% confluence and analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) with a luminometer. Lysates were analyzed in duplicate and cells lacking the ARE reporter were included as a control.

Mitochondrial ROS
Cells were incubated in serum-and phenol red-free DMEM with 5uM MitoSOX or 100nM Mitotracker Green FM (both Invitrogen) for 30 minutes and analyzed by flow cytometry. Mitochondrial ROS was plotted as the mitochondrial ROS signal (MitoSOX) normalized to mitochondrial mass (Mitotracker Green FM).

Nox Activity assay
Cells were washed twice with ice cold PBS, scraped in PBS, and centrifuged to obtain a cell pellet. The pellet was dounce homogenized in PBS + 1mM MgCl2 + 1mM EGTA + protease inhibitors and 100ul of homogenate was added to 100ul of 2× reaction buffer (PBS + 1mM MgCl2 + 1mM EGTA + 300mM sucrose + 10uM lucigenin [Sigma Aldrich] + 200uM NADPH [Sigma Aldrich]) and relative light units were measured with a luminometer every minute for 10 minutes. Specificity was confirmed by adding the specific Nox inhibitor diphenylene iodonium (10uM, Sigma Aldrich). Graphs show one representative measurement in the linear range (2-3 minutes) and are expressed as relative light units per μg protein.

Nrf2 stability
MEFs were treated with 25ug/ml cycloheximide (Acros Organics) for indicated time points and total cell lysates extracted and separated by SDS-PAGE. Gels were transferred to nitrocellulose (Biorad), and membranes were probed with Anti-Nrf2 antibody, affinity purified as described. 28

Quantification of proliferation and neoplasms
Values for proliferation were obtained by counting at least 5 fields of greater than 50 cells. For PanIN studies, only cells contained in PanIN-1a were included in the analysis (surrounding stromal, acinar or immune cells were all excluded). Proliferation is determined as a percentage and therefore represents the number of Ki-67 positive PanIN-1a cells /total PanIN-1a cells. For quantification of lung disease burden, total lung areas and neoplasm areas were calculated using Image J 29 and disease burden was represented as a percent of total area. For characterization of individual neoplasms, data was represented as neoplasms/ lung area. For quantification of PanIN per mouse, paraffin embedded pancreata were sectioned at 100 micron intervals and individual PanIN were counted.

Ras-GTP activity assays
Ras-GTP levels were determined according to the manufacturer's instructions (Millipore). Membranes were also blotted with anti-Rac antibody (Millipore) as a loading control.

Senescence-associated b-galactosidase staining
Cells were fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 minutes and stained in staining solution (Citric acid/Phosphate buffer pH 6.0, 150mM NaCl, 2mM MgCl, 5mM Potassium Ferricyanide, 5mM Potassium Ferrocyanide, and 1mg/ml X-gal) overnight at 37°C. Fields of at least 50 cells were counted, in triplicate. For tissue, fresh pancreas from mice harboring PanIN was cut into small pieces (approximately 1-3 mm cubes) and fixed for 2 hours in 4% PFA in PBS on ice, and incubated in staining solution overnight at 37°C. The pieces were then fixed in formalin overnight, followed by a quick processing and embedding in paraffin wax. Sections were cut with a thickness of 10 microns, dewaxed with minimal exposure to xylene, and counterstained with nuclear fast red. Five fields of at least 50 cells were counted per mouse.

Sequencing of KEAP1 and NRF2 in patient samples
Xenograft enriched pancreatic cancers were created as described previously 30 . Cell lines were obtained from the ATCC (Aspc-1, CAPAN1, CFPAC1, Hs766T, MiaPACA2, Panc-1 and Su86.86) or from other sources. PK8 and PK9 were kindly provided by Dr. A. Horii 31 . PL11, PL19, PL23, (kindly provided by Dr. E. Jaffee), and XPA1 (kindly provided by Dr. A. Maitra) were also used. Genomic DNA from immortalized peripheral blood lymphocytes of 29 patients with familial pancreatic cancer were obtained from the National Familial Pancreatic Tumor Registry (NFPTR). The acquisition of human tissues from resection specimens or the NFPTR was approved by the Institutional Review Board at the Johns Hopkins Hospital. The coding sequences of exons 2-6, encompassing the entire coding sequence of KEAP1, and exon 2 of NRF2 (official current gene name, NFE2L2), encompassing the KEAP1 binding domain, were amplified from genomic DNAs using intronic primers flanking each exon. Primers were designed using Primer3 online software (http://frodo.wi.mit.edu/primer3). PCR-amplified products were sequenced using nested primers and an ABI Prism model 3700 Applied Biosystems, Foster City, CA). Sequence analysis employed Sequencher™ version 4.8 software (Gene Codes, Ann Arbor, MI). Identified variants were verified by independent PCR amplification and reverse sequencing of the amplified products. The somatic or germline nature of each variant was determined by comparing their respective sequences to matched normal DNA available from the same patients. The following primers were used:

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  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.