Targeted deletion of Nrf2 reduces urethane-induced lung tumor development in mice

Abstract

Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2(+/+) and Nrf2(-/-) mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2(-/-) mice compared to Nrf2(+/+) mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2(-/-) mice than in Nrf2(+/+) mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2(+/+) mice relative to Nrf2(-/-) mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

Figure 1. Bronchoalveolar lavage analysis (BAL) and body weight changes at a pre-neoplastic stage.

(A) BAL analysis found significantly higher concentration of total protein and numbers of monocytes, lymphocytes, phagocytic macrophages, and epithelial cells in Nrf2^-/- mice at 9 and/or 11 wk after urethane treatment. Neutrophilic myeloperoxidase (MPO) activity in BAL fluids was also significantly higher in Nrf2^-/- mice than in Nrf2^+/+ mice. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2^+/+ mice. (B) Necrotic lung cell lysis and death was assessed by lactate dehydrogenase (LDH) activity in aliquots of BAL fluids using a colorimetric assay. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2^+/+ mice. (C) Percent whole body weight changes monitored during and after saline or urethane treatment. Mean±SD are presented (n = 9−24 in vehicle groups, n = 64−82 in urethane groups). *, p<0.05 Nrf2^+/+ saline-treated vs. urethane-treated mice (p<0.05). #, p<0.05 Nrf2 ^-/- saline-treated vs. urethane-treated mice. +, p<0.05 urethane-treated Nrf2^+/+ mice vs. urethane-treated Nrf2^-/- mice (p<0.05).

Figure 2. Early stage tumorigenesis at 12 wk.

(A) H&E-staining demonstrate pulmonary hyperplastic regions and early tumor development (arrow heads) 12 wk after the first urethane injection. Greater tumor cell proliferation in sporadically small adenomatous regions was found in Nrf2^+/+ mice than Nrf2^-/- mice as indicated by denser proliferating cell nuclear antigen (PCNA) localization in relatively more advanced tumors (inset). Representative light photomicrographs showing intermediate magnitude of pathology for each treatment group are presented (n = 3−8/group for H&E, n = 3/group for PCNA). AV, alveoli; TB, terminal bronchiole; BV, blood vessel; UN, uninvolved region. Arrow heads, tumor; arrows, PCNA-positive nuclei. Bars indicate 100 µm. (B) Numerous apoptotic airway cells in Nrf2^-/- mice were identified by TUNEL assay on paraffin-embedded lung tissue sections. TUNEL-positive nuclei of epithelial, endothelial, and smooth muscle cells on proximal lung sections were counted and normalized to airway surface (mm²) using digital image processing software. Few TUNEL-stained cells were found in vehicle control mice. Mean±SD are presented (n = 3/group). +, p<0.05 vs. urethane-treated Nrf2^+/+ mice. (C) Differential early tumor formation between Nrf2^+/+ and Nrf2^-/- mice. Average number of tumors (≥200 µm) per whole lung from each mouse was assessed in serial sections of paraffin-embedded lungs fixed with 10% NBF. Mean±SD are presented (n = 13−14/group). +, p<0.05 vs. urethane-treated Nrf2^+/+ mice.

Figure 3. Adenoma development and persistent lung inflammation at 22 wk.

(A) Average number of adenomas per whole lung from each animal (tumor multiplicity) and number of tumors per size ranges and average size (mm) of individual tumors were assessed in lungs fixed with Tellyesniczky's fixative. Mean±SD are presented (n = 4−5/group saline; n = 14/group for tumor multiplicity; n = 14−20/group for tumor size). This study was repeated once with similar n and responses determined. *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. urethane-treated Nrf2^+/+ mice. (B) Bronchoalveolar lavage (BAL) analysis of the number of neutrophils in Nrf2^+/+ and Nrf2^-/- mice at 22 wk after urethane treatment. Mean±SD are presented (n = 7−10/group urethane treated and n = 3−5/group for saline). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. urethane-treated Nrf2^+/+ mice.

Figure 4. Pulmonary Nrf2 expression and activation caused by urethane.

(A) Western blot analysis of total Nrf2 protein in uninvolved (UN) and tumor tissues compared to saline control in lung at 22 wk (57 kDa determined). Nuclear translocation of Nrf2 in urethane-treated lungs at 22 wk was determined by Western blot analysis in whole lung nuclear extracts (10 µg). Lamin B1 level was determined as an internal control. (B) Binding activity of nuclear protein (5 µg) to [γ³²P]ATP end-labeled oligonucleotide probe containing ARE consensus sequence was determined. Gel shift analysis demonstrated increased total ARE binding activity (arrows) of lung nuclear proteins after urethane treatment in Nrf2^+/+ mice. Supershifted bands (arrow head) indicate specific binding activity of nuclear Nrf2 or small Maf on ARE determined by addition of anti-Nrf2 or –Maf (F/G/K) antibodies to the reaction. Representative digitized bands of Western blotting and gel shift analysis (n = 2/group) are presented. (C) Immunohistochemistry for Nrf2 in saline- (a) or urethane-treated (c,e) tissue sections. Greater localization of Nrf2 proteins (brown dots) in growing adenomas and in conducting airway and alveolar epithelial cells was found in Nrf2^+/+ mice. Nrf2 was located mainly in airway epithelial cells of vehicle control lungs. Lung sections from Nrf2^-/- mice (e) are shown as a negative control for Nrf2. H&E-stained lower magnification of lung sections treated with vehicle (b) or urethane (d,f) are depicted for general histology. Representative images showing intermediate degree of Nrf2 staining for each treatment group are presented (n = 3/group). AV = alveoli, BR = bronchi or bronchiole, TB = terminal bronchiole, BV = blood vessel, Bar = 100 µm.

Figure 5. Lung gene expression profiles during urethane-induced tumorigenesis.

(A) Urethane effects on gene expression during lung tumorigenesis were compared by biological function and disorder categories of genes significantly changed in pre-/early-neoplastic stage at 12 wk (top), in uninvolved tissues (UN) at 22 wk (middle), and in tumors at 22 wk (bottom) using Ingenuity Pathway Analysis (IPA). Top-ranked categories are depicted against -log(p) determined by IPA. (B) Among the lung gene transcripts significantly (p<0.05) changed in uninvolved (UN, n = 1564) and tumor (n = 8683) tissues compared to saline-treated lung tissues, Venn diagram analysis identified genes changed in either tissue or in common at 22 wk after the initial urethane treatment (top). Expression profiles of all tumor genes (n = 8683, 22 wk UT-Tum) are depicted in pre-/early neoplastic microenvironment (12 wk) and in UN tissues of tumor-bearing lungs at 22 wk (bottom). Color bar indicates expression intensity of individual transcripts normalized to their time-matched saline control levels expressed in yellow (yellow to blue, down-regulation; yellow to red, up-regulation). Y axis = log₂ (normalized average intensity), Sal = saline-treated whole lung, UT = urethane-treated (whole lung for 12 wk, UN or Tum for 22 wk), UN = uninvolved tissue, Tum = tumor tissue.

Figure 6. Effect of Nrf2 deletion on gene expression during lung tumorigenesis.

(A) Biological function and disorder categories of the Nrf2-dependent genes (i.e. differentially expressed between Nrf2^+/+ and Nrf2^-/- mice) significantly changed in pre-/early neoplastic stage at 12 wk (top) and in tumors at 22 wk (bottom) were analysis by Ingenuity Pathway Analysis (IPA). Top-ranked [-log(p)] categories are depicted and compared between the two tumorigenesis stages. (B) A total of 118 gene transcripts varied significantly (p<0.05) between Nrf2^+/+ and Nrf2^-/- mice 12 wk after urethane treatment were grouped into 4 expression profiles (set 0–3). Expression level of each transcript was normalized to corresponding Nrf2^+/+ saline controls and indicated as relative log ratio [log₂ (normalized average intensity)]. Genes are listed in Dataset 1 and in Table 1. Sal = saline-treated lung, UT = urethane-treated lung. (C) Lung tumor genes (n = 376) significantly (p<0.05) different between Nrf2^+/+ and Nrf2^-/- mice at 22 wk after urethane treatment are clustered into 4 expression profile groups (set 0–3). Expression level of each gene was normalized to that of Nrf2^+/+ saline controls and indicated as relative log ratio [log₂ (normalized average intensity)]. Genes are listed in Dataset 2 and in Table 2. Sal = saline-treated lung, UN = uninvolved tissue, Tum = tumor tissue. (D) Among the Nrf2-dependently changed genes in pre-/early neoplastic stage at 12 wk (n = 118) and in tumors at 22 wk (n = 376), Venn diagram analysis identified gene transcripts that were changed at either time point (12 and 22 wk) and/or were genes in common between the two time points (i.e. 12 genes).

Figure 7. Proposed role for Nrf2 in urethane-induced lung tumorigenesis.

Urethane treatment causes pulmonary inflammation and injury during the pre-neoplastic stage, which results in overproduction of reactive oxygen species (ROS) and cellular death by necrosis and apoptosis. Compared to Nrf2 ^+/+ mice, Nrf2^-/- mice have lowered cell survival factors including cellular redox and drug metabolism enzymes (e.g., glutathione synthetase, UDP glucuronosyl transferase 1 family) and cell maintenance systems including numerous metabolic enzymes and transport proteins (e.g., solute carrier family). These mice therefore have heightened cellular destruction factors (e.g., ROS, airway secretion, inflammation), which overwhelms cellular cytoprotection tools and causes mass death of injured cells including tumor initiated cells during the pre-neoplastic stage. Nrf2^-/- mice also exhibit dysregulated expressions of many genes involved in cell cycle and death (e.g., CDC28 protein kinases, cyclin D1, cyclin dependent kinase inhibitor 2C, B-cell leukemia/lymphoma 6, unc-119 homolog) relative to wild type mice during tumorigenesis. Overall, increased susceptibility to acute injury due to lack of survival signals leading to net cell loss is beneficial to Nrf2^-/- mice for their tumor suppression.