USP4-mediated deubiquitination of NOTCH1 inhibits HNSCC progression through regulating oxidative stress and ferroptosis

The discovery of new drugs and focused treatment strategies is significantly hampered by a lack of understanding of the complex pathophysiology of head and neck squamous cell carcinoma (HNSCC). Unbalances in the oxidative stress and anti-oxidative response pathways are important risk factors for the development of HNSCC. Tumor growth can be inhibited and significant clinical improvements can be obtained for Cancer patients by therapeutic approaches that target the Ubiquitin-Specific Protease 4 (USP4) and its targeted response element in response to oxidative stress. To evaluate USP4 expression and its prognostic significance, this study examined clinical HNSCC specimens as well as data from The Cancer Genome Atlas (TCGA), The International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO). To find the main differential gene, proteomics assay and RNA-seq Sequencing were performed using the 4-nitroquinoline N-oxide (4-NQO)- or N-nitroso-N-methyl-4-aminobutyric acid (NMBA)-induced in vitro and in vivo paradigm. Additionally, the impact of USP4 and its downstream events on HNSCC progression was assessed using mice with 4-NQO- or NMBA-mediated HNSCC models that were either epithelial-specific USP4 deletion or restoration mice. Additionally, we used qPCR, ELISA, and western blotting to evaluate the levels of USP4-related signaling events. Ki67 immunofluorescence and transwell assays were used to assess cell invasion, migration, and proliferation. Finding out if USP4 is a Ubiquitin-Specific Protease that regulates oxidative stress, which stabilizes NOTCH1 and deactivates the anti-oxidative transcription-related pathway, as well as examining USP4's role in HNSCC development. We observed that USP4 is a protein that interacts with NICD1 and catalyzes the deubiquitination of NICD1 in HNSCC cells, making it a significant endogenous stabiliser of NOTCH1 intracellular domain (NICD1) activity. Epithelial USP deletion destabilizes NICD1 and abrogates NOTCH1 signaling. This upregulates antioxidant genes, blocks lipid peroxidation-induced Ferroptosis, and eventually accelerates HNSCC progression. However, transgenic overexpression-mediated USP4 gene therapy decreases the development of Cancer in animal models treated with 4-NQO. Mechanistically, USP4 raises NICD1 abundance and activates its downstream signaling cascade by binding to NICD1 in response to oxidative stress, deconjugating ubiquitination chains. Additionally, in vivo evidence reveals that loss of USP4 accelerates HNSCC progression by destabilizing NICD1 and inhibiting Ferroptosis. Crucially, decreased expression of USP4, which is connected to the severity of HNSCC, indicates a poor prognosis. Our study demonstrated that oxidative stress and Ferroptosis participated in HNSCC progression by regulating USP4 axis, leading to deactivation and degradation of NOTCH1 pathway. These findings suggest that USP4 may be a good therapeutic target for HNSCC therapy.