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Updated: Jul 16, 2025
Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC
Nathan T Wamsley1, Emily M Wilkerson1, Li Guan2
1Department of Cell Biology and Physiology, Washington University in St Louis, St Louis, Missouri, USA.
Abstract:
The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.
Insights
This study developed a rapid proteomics assay to measure NRF2 pathway proteins in head and neck cancer. This assay can stratify patients by NRF2 activity, potentially guiding treatment decisions for better outcomes.
Area of Science:
- Oncology
- Proteomics
- Biomarker Discovery
Background:
- The NFE2L2 (NRF2) pathway promotes cancer progression, immune evasion, and treatment resistance.
- Stratifying patients by NRF2 activity could optimize head and neck squamous cell carcinoma (HNSCC) treatment.
- A need exists for a clinically actionable assay to quantify NRF2 pathway components in HNSCC.
Purpose of the Study:
- To develop and validate a mass spectrometry-based targeted proteomics assay for quantifying NRF2 pathway proteins and clinical biomarkers in HNSCC.
- To improve the SureQuant algorithm for enhanced throughput, sensitivity, and precision in proteomic analysis.
- To assess the clinical utility of the assay in HNSCC patient samples and cancer cell models.
Main Methods:
- Development of a targeted proteomics assay using internal standard-triggered parallel reaction monitoring (PRM) to quantify 69 NRF2 pathway components and 21 clinical proteins.
- Optimization of the SureQuant PRM acquisition algorithm for improved performance.
- Application of the assay to 27 cancer cell models and formalin-fixed paraffin-embedded (FFPE) HNSCC tissues.
Main Results:
- The optimized assay quantified 35 NRF2-responsive proteins in cancer cell models.
- In HNSCC tissues, NRF2 signaling intensity correlated with NRF2-activating mutations and SOX2 expression.
- Biomarkers for T-cell infiltration and human papillomavirus (HPV) infection status were identified and correlated.
- The assay demonstrated high throughput, quantifying over 600 peptides from FFPE tissues in under 90 minutes.
Conclusions:
- A clinically actionable HNSCC protein biomarker assay was established, enabling rapid quantification of NRF2 pathway components and clinical markers.
- The assay facilitates patient stratification based on NRF2 activity, potentially guiding personalized treatment strategies.
- This proteomic approach provides valuable insights into HNSCC biology, including immune evasion and viral activity.
