Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines
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Izabela N F Gomes1, Renato J da Silva-Oliveira1,2, Luciane Sussuchi da Silva1
1Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784-400, Brazil.
Abstract:
Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.
Insights
Researchers developed a cetuximab-resistant head and neck squamous cell carcinoma (HNSCC) model. Overexpression of KRAS, RhoA, and CD44 correlated with resistance, suggesting new biomarkers for cetuximab therapy in HNSCC.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Cetuximab is an FDA-approved anti-EGFR antibody for head and neck squamous cell carcinoma (HNSCC).
- Predictive biomarkers for cetuximab response in HNSCC are currently unknown.
- Understanding resistance mechanisms is crucial for improving HNSCC treatment.
Purpose of the Study:
- To investigate the molecular mechanisms of cetuximab resistance in HNSCC.
- To identify potential biomarkers for predicting cetuximab response.
- To explore strategies for overcoming anti-EGFR resistance in HNSCC.
Main Methods:
- Established an in vitro cetuximab-resistant HNSCC cell line (FaDu).
- Performed cell viability, functional assays, Western blot, and human cell surface protein analysis.
- Utilized whole-exome sequencing (WES) and NanoString for mutational profiling and copy number alterations (CNA).
- Analyzed differential gene expression associated with resistance pathways.
Main Results:
- Resistant cells showed significantly higher IC50 values compared to parental cells.
- WES identified mutations in cancer-related genes; 36 differentially expressed genes were linked to resistance pathways (EGFR, RAS, MAPK, mTOR).
- Overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance.
- Resistant cells exhibited increased adhesion, colony formation, and migration, indicating an aggressive phenotype.
- EGFR phosphorylation was inhibited, while mTOR signaling increased in resistant cells.
Conclusions:
- Identified KRAS, RhoA, and CD44 overexpression as potential biomarkers for cetuximab resistance in HNSCC.
- mTOR and RhoA overexpression may serve as novel predictive biomarkers for cetuximab response.
- These findings suggest new therapeutic strategies to overcome anti-EGFR resistance in HNSCC.
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