NRF2 signaling plays an essential role in cancer progression through the NRF2-GPX2-NOTCH3 axis in head and neck squamous cell carcinoma

Abstract Background: The activation of nuclear factor erythroid 2–related factor 2 (NRF2) has been observed in various cancers. Yet its exact contribution to the development of head and neck squamous cell carcinoma (HNSCC) remains undetermined. Methods: We systematically investigated the role of NRF2 in HNSCC, ultimately selecting GPX2, which exhibited a marked downregulation, for a detailed mechanistic analysis. Firstly, we knocked out NRF2 by CRISPR-Cas9, and subsequently confirmed by RT-qPCR and Western Blot. The role of NRF2was evaluated through various assays, including cell growth assays, colony formation assays, 3D cultures, cell migration and invasion assessments, ROS detection, and xenograft tumor models. Furthermore, we performed RNA sequencing on NRF2-KO cells compared to NRF2-WT cells identified GPX2 as a downstream target of NRF2. This finding led us to examine the role of GPX2 in the maintenance of cancer stem cells (CSCs). Notably, CSC analysis indicated the involvement of the NOTCH signaling pathway in HNSCC progression, and the critical role of NOTCH3 was confirmed using the serious experimental approaches mentioned earlier. Results: We previously found that NRF2 signaling is critical for the differentiation of squamous basal progenitor cells, while disruption of NRF2 causes basal cell hyperplasia. In this study, we revealed a correlation between elevated NRF2 activity and poor outcomes in HNSCC patients. We demonstrated that NRF2 facilitates tumor proliferation, migration, and invasion, as evidenced by both in vitro and in vivo studies. Significantly, NRF2 augments the expression of the antioxidant enzyme GPX2, thereby enhancing the proliferative, migratory, and invasive properties of HNSCC cells. Activation of GPX2 is critical for sustaining CSCs by up-regulating NOTCH3, a key driver of cancer progression. Conclusions: NRF2 regulates HNSCC progression through the NRF2-GPX2-NOTCH3 axis. Our findings proposed that pharmacological targeting of the NRF2-GPX2-NOTCH3 axis could be a potential therapeutic approach against HNSCC.