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Modelling HNSCC in mice with hyperactivated NRF2 pathway

ABSTRACT: Purpose: Alterations in the KEAP1/NFE2L2 (NRF2)/CUL3 pathway occur in ~20% of human head and neck squamous cell carcinomas (HNSCC) and are associated with resistance to standard-of-care therapy. However, this pathway's role in radiotherapy resistance in HNSCC has not been well-studied. Experimental Design: We generated genetically engineered mouse models and developed primary murine cancer cell lines harboring mutations commonly observed in human HNSCC, including inducible activation of PIK3CA and deletion of Trp53, with or without Keap1 loss. Primary tumors were initiated via 4-hydroxytamoxifen injection ± the tobacco carcinogen benzo[a]pyrene (BAP) into the oral buccal mucosa. Tumors were analyzed using Western blotting, immunohistochemistry, RNA sequencing (RNA-seq), and subjected to fractionated radiation therapy to investigate the role of the KEAP1/NRF2 pathway in radioresistance and modulation of the tumor immune microenvironment. Results: BAP exposure accelerated primary tumor formation within one month, with histological analysis confirming invasive squamous cell carcinoma, validated by cytokeratin and differentiation marker expression. Primary cell lines derived from Keap1-haploinsufficient tumors exhibited upregulation of NRF2 target genes and a radioresistant phenotype, which was reversed post-Nrf2 knockdown in vitro. Bulk RNA-seq revealed that Keap1 haploinsufficiency correlated with NRF2 pathway activation, increased myeloid infiltration, and enhanced angiogenic signatures. In vivo, Keap1 haploinsufficiency promoted accelerated tumor growth and decreased survival. Finally, using fractionated radiotherapy, we showed that Keap1 haploinsufficient primary tumors were significantly more radioresistant than Keap1-proficient tumors, regardless of BAP exposure. Conclusion: These data demonstrate that Keap1 haploinsufficiency in HNSCC is linked to unfavorable tumor immune microenvironment, aggressive growth, and a radioresistant phenotype.

ORGANISM(S): Mus musculus

PROVIDER: GSE301971 | GEO | 2025/07/08

REPOSITORIES: GEO

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Project description:Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1) sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp) genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S -transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification. We used microarrays to detail the global programme of gene expression in response to Nrf2 activation and identified distinct classes of up- and down-regulated genes. process. Gene expression in livers of Nrf2-null, WT, Keap1-KD, and Keap1-HKO mice was determined using Affymetrix Mouse 430.20 arrays by the KUMC Microarray Core Facility. Biological cRNA replicates (n=3) of each genotype were hybridized to an individual array.

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Modelling HNSCC in mice with hyperactivated NRF2 pathway

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