Edited by Xiao-Fan WangMutations in the genes encoding nuclear factor (erythroidderived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2 ؊/؊ , Keap1 ؊/؊ , K5Cre;Pkm2 fl/fl ;Keap1 ؊/؊ , and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro. In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2 high ESCC in humans.Esophageal cancer affects 16,940 adults in the United States, and the 5-year survival rate is 18% (1). There are mainly two histological types of esophageal cancer, squamous cell carcinoma (ESCC) 4 and adenocarcinoma, each having a distinct etiology. Low income, moderate/heavy alcohol intake, tobacco use, and infrequent consumption of raw fruits and vegetables account for almost all cases of ESCC (2). With the recent technological advances in NextGen sequencing, human ESCC samples from North and South America, China, Japan, Vietnam, and Malawi have been sequenced. Among many gene mutations, nuclear factor (erythroid-derived 2)-like 2 (NRF2 or NFE2L2) mutations are commonly seen with a frequency over 5%, even up to ϳ20% in certain reports. Mutations in other genes of the NRF2 signaling pathway, Kelch-like ECH associated protein 1 (KEAP1) and cullin 3 (CUL3), are relatively less common. NRF2 mutations are mostly located in the DLG and ETGE motifs (KEAP1-binding domain) and the DNA-binding domain, whereas KEAP1 mutations tend to be scattered across the whole gene. NRF2 mutations and KEAP1 mutations tend to be mutually exclusive (3,4).As a major cellular defense mechanism, the NRF2 signaling pathway is known to regulate expression of enzymes involved in detoxification and ant...

Esophageal squamous cell carcinoma (ESCC) is a deadly disease that requires extensive research. Here, we review the current understanding of the functions of the nuclear factor erythroid-derived 2-like 2 (NRF2) signaling pathway in the esophagus. Genomic data suggest that gene mutations and several other mechanisms result in NRF2 hyperactivation in human ESCC. As a consequence, NRF2 ESCC is more resistant to chemoradiotherapy and associated with poorer survival than NRF2 ESCC. Mechanistically, we believe NRF2, functioning as a transcription factor, causes an esophageal phenotype through regulation of gene transcription. We discuss metabolism, mitochondria, proteasomes, and several signaling pathways as downstream players that may contribute to an esophageal phenotype due to NRF2 hyperactivation. Finally, strategies are proposed to target the NRF2 signaling pathway for therapy of NRF2 ESCC.

Oxidative stress is known to play a pivotal role in the development of oral squamous cell carcinoma (OSCC). We have demonstrated that activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway has chemopreventive effects against oxidative stress-associated OSCC. However, Nrf2 have dual roles in cancer development; while it prevents carcinogenesis of normal cells, hyperactive Nrf2 also promotes the survival of cancer cells. This study is aimed to understand the function of Nrf2 in regulating cellular behaviors of OSCC cells, and the potential mechanisms through which Nrf2 facilitates OSCC. We established the Nrf2-overexpressing and Nrf2-knockdown OSCC cell lines, and examined the function of Nrf2 in regulating cell proliferation, migration, invasion, cell cycle and colony formation. Our data showed that Nrf2 overexpression promoted cancer phenotypes in OSCC cells, whereas Nrf2 silencing inhibited these phenotypes. In addition, Nrf2 positively regulated Notch signaling pathway in OSCC cells in vitro. Consistent with this observation, Nrf2 activation in Keap1−/− mice resulted in not only hyperproliferation of squamous epithelial cells in mouse tongue as evidenced by increased expression of PCNA, but also activation of Notch signaling in these cells as evidenced by increased expression of NICD1 and Hes1. In conclusion, Nrf2 regulates cancer behaviors and Notch signaling in OSCC cells.

Background: The Nrf2/Keap1 pathway is critical for human cells to respond to oxidative stress. In basal conditions the Keap1 homodimer is bound to Nrf2 in the cytoplasm. When stressed, Keap1 releases Nrf2 which translocates into the nucleus, binds the antioxidant response element, and activate antioxidative genes. Somatic mutations of Nrf2 or Keap1 gene can cause constitutive activation of this pathway, and thus results in chemoradioresistance and poor survival in patients with esophageal squamous cell carcinoma (ESCC). Our goal is to identify chemical Nrf2 inhibitors for targeted therapy of Nrf2high ESCC in the future. Methods: Possible Nrf2 inhibitors were identified using high throughput screening of 1,280 compounds in the Prestwick library, and 35,000 compounds in the Asinex library with Nqo1-EGFP H1299 cells stimulated by an Nrf2 activator (CDDO). Nrf2 activity was monitored via Incucyte by measuring the total normalized green fluorescence divided by the total normalized red fluorescence (tagged nuclear cherry marker). The red channel assessed cell viability and cytotoxicity of compounds. Inhibitors were identified if they met a threshold of >50% inhibition. Lead compounds will be further validated in vitro with KYSE 450 (Nrf2low+Keap1 knockdown) and KYSE 70 (Nrf2high) by examining their effects on expression of nuclear Nrf2 and its downstream target gene (Nqo1). These compounds will also be validated in vivo using Sox2CreER;Nrf2Ki/Ki mice. Results: We successfully established the in vitro assay with Nqo1-EGFP H1299 cells for high throughput screening of Nrf2 inhibitors. Eight lead compounds were identified from the Prestwick library and validated as Nrf2 inhibitors via a dose-response experiment (IC50<1μM). Screening of the Asinex library and further validations are still ongoing. Conclusion: We have identified Nrf2 inhibitors through high-throughput screening and will test them in vitro and finally in vivo. Citation Format: Seth Weir, Xiaoxin Chen, Chorlada Paiboonrungruan. Identification and validation of Nrf2 inhibitors in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5856.