Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines

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.

KRAS mutations upregulate Runx1 to promote occurrence of head and neck squamous cell carcinoma

“…In addition, germline mutations occur in the 3' untranslated region (3' UTR) of KRAS with an incidence of 15%–32% in HNSCC. Both KRAS abnormalities above can lead to poor prognosis and cetuximab resistance 17,21,22 . However, the role of KRAS mutation in HNSCC is still unclear, and it is of great significance to explore new mechanisms and identify novel therapeutic targets for KRAS ‐mutated HNSCC patients with poor prognosis.…”

Unveiling the role of MGMT and DAPK hypermethylation in response to anti‐EGFR agents: Molecular insights for advancing HNSCC treatment

“…Even in cases where an initial positive response occurs, patients eventually develop or acquire secondary resistance to these inhibitors, resulting in relapse after several months 19,20 . Some studies tried to unveil the complex mechanisms of resistance, yet, this knowledge has not translated to effective patient treatment 21 . Consequently, resistance to anti‐EGFR inhibitors has become a significant clinical concern, emphasizing the importance of exploring new EGFR inhibitors for the treatment of HNSCC tumors.…”

“…19,20 Some studies tried to unveil the complex mechanisms of resistance, yet, this knowledge has not translated to effective patient treatment. 21 Consequently, resistance to anti-EGFR inhibitors has become a significant clinical concern, emphasizing the importance of exploring new EGFR inhibitors for the treatment of HNSCC tumors. Clinical trials with agents such as Afatinib and Allitinib hold promise in addressing this issue.…”

Genome-Wide Super-Enhancer-Based Analysis: Identification of Prognostic Genes in Oral Squamous Cell Carcinoma

“…Various analytical approaches (gene expression profiles, noncoding RNA profiles, and chromosomal alteration) have been explored to elucidate the molecular mechanisms of drug resistance [ 8 , 9 ]. Previous studies have revealed that the expression of anticancer drug excretion genes, DNA repair genes, antiapoptotic genes, and epithelial mesenchymal transition (EMT)-related genes may be activated in anticancer drug-resistant cancer cells [ 10 , 11 , 12 , 13 ].…”