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Abstract Introduction: Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a key role in protecting cells from oxidative stress. NRF2 is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1), which facilitates NRF2 ubiquitination and proteosomal degradation under homeostatic conditions. NRF2 activating mutations occur at a frequency of 5. 9% in muscle-invasive bladder cancer (MIBC). However, the role of NRF2 in MIBC has not been fully understood. The aim of this study is to clarify how NRF2 affect to the normal epithelial cells and the tumor cells in bladder. Methods: We used genetically engineered mice (GEM) with a Cre inducible Nrf2 activating mutation (LSL-Nrf2-E79Q). To evaluate how Nrf2 affects the tumorigenesis process in vivo, the N-butyl-N- (4-hydroxybutyl) -nitrosamine (BBN) was administered to Upk3a-CreERT2; LSL-Nrf2-E79Q/+ (UN) mice. We also established normal urothelial organoids and BBN tumor derived organoids from wild-type (WT) and Krt5-CreERT2; LSL-Nrf2-E79Q/+ (K5N) mice for ex vivo study. To induce overexpression of NRF2, we utilized KI696 (KEAP1-NRF2 inhibitor) as a pharmacological agent and performed NAD/NADH assay to quantify the reductive stress. Additionally, we performed single cell RNA sequencing (scRNA-seq) with WT, UN and K5N mice bladder to identify the effect of Nrf2E79Q on the normal epithelial cells. Results: In BBN induced tumor model in vivo, UN mice did not differ in time to tumor development from the control WT mice. Furthermore, UN tumors had lost the post-recombination allele of LSL-Nrf2-E79Q in genomic PCR, meaning cells expressing Nrf2E79Q are selected against during the process. In ex vivo study, KI696 induced reductive stress in both WT normal urothelial organoids and BBN tumor derived organoids, but inhibited cell proliferation only in normal urothelial organoids. In K5N organoids, Nrf2E79Q activation enhanced relative mRNA expression of a number of basal related genes. In addition to that, vehicle organoids in differentiation media showed phenotypic differentiation, whereas those treated with tamoxifen did not demonstrate phenotypic signs of differentiation suggesting Nrf2 activation promotes a basal-like phenotype. Furthermore, we annotated three clusters in mice bladder epithelial cells as luminal, intermediate and basal using scRNA-seq and looked at their proportion among WT, UN and K5N mice. We found that the percentage of luminal cells was decreased and intermediate and basal cells increased in both UN and K5N mice compared to WT mice. This indicated that Nrf2E79Q promotes disadvantage in luminal cells in UN mice, whereas it contributes to the increase in intermediate and basal cells in K5N mice. Conclusion: These results suggested that Nrf2E79Q promotes a fitness disadvantage in normal mice urothelium. Furthermore, Nrf2E79Q inhibits differentiation of K5N organoids and drives a basal-like phenotype in GEM models. Further investigation is needed to clarify the mechanism of NRF2 activating mutation contributing the tumorigenesis in bladder. Citation Format: Akihiro Hamada, Yuki Kita, Di Wu, Mi Zhou, Sean Clark-Garvey, Andrew Gdowski, Michael Sturdivant, Wolfgang Beckabir, Elliott Drew Toomer, Emily Kounlavong, Lucia Lim, Kathryn Gessner, Jeffrey Damrauer, William Kim. NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype abstract. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30 (10Suppl): Abstract nr A016.
Hamada et al. (Fri,) studied this question.