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The transcription factor Nrf2 activates transcription of cytoprotective genes during oxidative and electrophilic insults. Nrf2 activity is regulated by Keap1 in a stress-dependent manner in normal cells, and somatic loss-of-function mutations of Keap1 are known to induce constitutive Nrf2 activation, especially in lung adenocarcinomas, conferring survival and proliferative benefits to tumors. Therefore, several therapeutic strategies that aim to inhibit Nrf2 in tumors have been developed for the treatment of Nrf2-activated cancers. Here we addressed whether targeting Nrf2 activation in the microenvironment can suppress the progression of Nrf2-activated tumors. We combined two types of Keap1-flox mice expressing variable levels of Keap1 with a Kras-driven adenocarcinoma model to generate Keap1-deficient lung tumors surrounded by normal or Keap1-knockdown host cells. In this model system, activation of Nrf2 in the microenvironment prolonged the survival of Nrf2-activated tumor-bearing mice. The Nrf2-activated microenvironment suppressed tumor burden; in particular, preinvasive lesion formation was significantly suppressed. Notably, loss of Nrf2 in bone marrow–derived cells in Nrf2-activated host cells appeared to counteract the suppression of Nrf2-activated cancer progression. Thus, these results demonstrate that microenvironmental Nrf2 activation suppresses the progression of malignant Nrf2-activated tumors and that Nrf2 activation in immune cells at least partially contributes to these suppressive effects.Significance:This study clarifies the importance of Nrf2 activation in the tumor microenvironment and in the host for the suppression of malignant Nrf2-activated cancers and proposes new cancer therapies utilizing inducers of Nrf2.

Figure S1: Expression of Keap1 and Nqo1 in Keap1WT/WT, Keap1FB/FB and Keap1FA/FA mouse tissues; Figure S2: Analyses of tumor-bearing mouse cohorts; Figure S3: Origins of tumor cells in Kras::Keap1WT/WT, Kras::Keap1FB/FB and Kras::Keap1FA/FA lungs; Figure S4. Analyses of tumor-bearing Kras::Keap1FA/FA mouse cohorts that received Keap1FA/FA, Keap1WT/WT or Nrf2-/- bone marrows; Table S1: Primers and probes for qRT-PCR analyses; Table S2: Primers for analyses of recombination efficiency; Supplementary Materials and Methods (Establishment of mouse embryonic fibroblasts (MEFs), Validation of the recombination efficiency, Immunoblot analyses, Histology and Immunohistochemistry, Flow cytometry, and isolation of CD8+ T cells in tumor-bearing lungs)

Nrf2 activates cytoprotective gene expression, and Nrf2 activity is regulated through at least two protein degradation pathways: the Keap1-mediated and β-TrCP-mediated pathways. To address the relative contributions of these pathways, we generated knock-in mouse lines expressing an Nrf2(SA) mutant that harbored two substitution mutations of serine residues interacting with β-TrCP. The homozygous (Nrf2(SA/SA)) mice grew normally, with Nrf2 levels comparable to those of wild-type (WT) mice under unstressed conditions. However, when Keap1 activity was suppressed, high levels of Nrf2 accumulated in Nrf2(SA/SA) macrophages compared with that in WT macrophages. We crossed Nrf2(SA/SA) mice with mice in which Keap1 was knocked down to two different levels. We found that the Nrf2(SA/SA) mutation induced higher Nrf2 activity when the Keap1 level was strongly reduced, and these mice showed severe growth retardation. However, activation and growth retardation were not evident when Keap1 was moderately suppressed. These increases in Nrf2 activity induced by the Nrf2(SA) mutation caused severe hyperplasia and hyperkeratosis in the esophageal epithelium but did not cause abnormalities in the other tissues/organs examined. These results indicate that the β-TrCP-mediated pathway cooperates with the Keap1-mediated pathway to regulate Nrf2 activity, which is apparent when the Keap1-mediated pathway is profoundly suppressed.

The transcription factor Nrf2 activates transcription of cytoprotective genes during oxidative and electrophilic insults. Nrf2 activity is regulated by Keap1 in a stress-dependent manner in normal cells, and somatic loss-of-function mutations of Keap1 are known to induce constitutive Nrf2 activation, especially in lung adenocarcinomas, conferring survival and proliferative benefits to tumors. Therefore, several therapeutic strategies that aim to inhibit Nrf2 in tumors have been developed for the treatment of Nrf2-activated cancers. Here we addressed whether targeting Nrf2 activation in the microenvironment can suppress the progression of Nrf2-activated tumors. We combined two types of Keap1-flox mice expressing variable levels of Keap1 with a Kras-driven adenocarcinoma model to generate Keap1-deficient lung tumors surrounded by normal or Keap1-knockdown host cells. In this model system, activation of Nrf2 in the microenvironment prolonged the survival of Nrf2-activated tumor-bearing mice. The Nrf2-activated microenvironment suppressed tumor burden; in particular, preinvasive lesion formation was significantly suppressed. Notably, loss of Nrf2 in bone marrow–derived cells in Nrf2-activated host cells appeared to counteract the suppression of Nrf2-activated cancer progression. Thus, these results demonstrate that microenvironmental Nrf2 activation suppresses the progression of malignant Nrf2-activated tumors and that Nrf2 activation in immune cells at least partially contributes to these suppressive effects. Significance: This study clarifies the importance of Nrf2 activation in the tumor microenvironment and in the host for the suppression of malignant Nrf2-activated cancers and proposes new cancer therapies utilizing inducers of Nrf2.

Sickle cell disease (SCD) is a recessively inherited blood disorder caused by abnormal β-globin production. The β-globin mutation changes erythrocyte morphology into a sickle shape and increases erythrocyte vulnerability to hemolysis. Oxidative stress and concomitant inflammation eventually result in damage to multiple organs. Nrf2 is a master regulator of the oxidative stress response, homeostasis, and metabolism. Keap1 modulates Nrf2 protein levels; Nrf2 inducers alter nuclear Nrf2 levels by interacting with Keap1. Genetic modification of Keap1 helps to reduce inflammation and tissue damage in SCD model mice through Nrf2 induction. Here, we investigated the benefits of a mild and safe Nrf2 agonist, sulforaphane (SFN), in ameliorating SCD pathology in a murine model. SFN is a phytochemical and is found in cruciferous vegetables as its inert precursor, glucoraphanin. We found that dietary SFN administration for 14 days or 2 months increased the expression of Nrf2-dependent cytoprotective genes, but SFN uptake did not have deleterious effects on the food consumption and growth of SCD model mice. SFN ameliorated the liver damage of SCD mice, which could be validated by the rescue of liver function and the significantly reduced liver necrotic area. SFN administration also helped to eliminate heme released from lysed sickle cells. These results indicate that dietary supplementation with SFN relieves SCD symptoms by inducing Nrf2 and support our contention that SFN is a potential drug for the long-term treatment of children with SCD.