Publication Date

10-1-2025

Journal

Clinical Cancer Research

DOI

10.1158/1078-0432.CCR-25-0112

PMID

40742317

PMCID

PMC12428276

PubMedCentral® Posted Date

9-13-2025

PubMedCentral® Full Text Version

Author MSS

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 by Western blotting, IHC, and RNA sequencing and subjected to fractionated radiotherapy 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 1 month, with histologic 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 after Nrf2 knockdown in vitro. Bulk RNA sequencing 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.

Conclusions: These data demonstrate that Keap1 haploinsufficiency in HNSCC is linked to unfavorable tumor-immune microenvironment, aggressive growth, and a radioresistant phenotype.

Keywords

NF-E2-Related Factor 2, Animals, Mice, Radiation Tolerance, Humans, Squamous Cell Carcinoma of Head and Neck, Kelch-Like ECH-Associated Protein 1, Head and Neck Neoplasms, Tumor Microenvironment, Cell Line, Tumor, Disease Models, Animal, Gene Expression Regulation, Neoplastic, Signal Transduction

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