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.

Keywords: EGFR; biomarkers; cetuximab; drug resistance; head and neck tumors; in vitro; pre-clinical.

Figure 1

Cetuximab resistant model establishment and characterization. (A) EGFR signaling of parental and cetuximab-resistant clones after cetuximab resistant model establishment. (B) Cell viability assay of parental and resistant clone upon cetuximab exposition in 72 h. (C) Cell morphology of parental and resistant cells P: FaDu parental; R: FaDu resistant; C: Clone. The images were acquired in 10× magnification.

Figure 2

Karyotypes and Copy number alterations (CNA) overview of FaDu parental and FaDu resistant cells. (A) Representative metaphase of FaDu parental and FaDu resistant cells. (B) Overview of the CNAs found in FaDu resistant cells in comparison with FaDu parental cells. In red gain and in blue deletions. M: marker chromosome.

Figure 3

Molecular characterization after cetuximab-acquired resistance. (A) Heatmap of genes altered in FaDu parental and FaDu resistant cells. In red are represented the overexpressed genes, and in blue the downregulated genes. FaDu parental is shown in purple and FaDu resistant in green. (B) Genetic interaction network associated with cetuximab resistance on the STRING database. In this figure, each circle represents a protein (node), and each connection represents a direct or indirect connection (edge). Line color indicates the type of interaction evidence: purple—experimental evidence, light blue—curate database, black—co-expression, pink—experimentally determined, yellow—text mining, dark blue—gene co-occurrence (MAPK associated genes are shown in red, RAS associated genes are shown in blue, and mTOR signaling-related genes are shown in yellow. p. adjusted <0.01; FC ≥ 2.

Figure 4

Cell surface markers and cytokines profile expression in FaDu parental and FaDu resistant cells. (A) Representation of cell-surface markers expression in FaDu parental and FaDu resistant cells. p. adjusted < 0.01. (B) Representative images of Cytokines protein array in FaDu parental and FaDu resistant cells. (C) Bars demonstrated the cytokines differential expression in FaDu cells.

Figure 5

EGFR-FITC+ internalization and mTOR-FITC+ expression in FaDu parental and FaDu resistant cells. (A) Subcellular protein fractionation assay for EGFR detection in parental and resistant cells. (B) p-EGFR nuclear translocation in resistant cells by Immunofluorescence assay. (C) p-mTOR immunofluorescence assay in parental and resistant cells. (D) mTOR-related genes differentially expressed in FaDu parental and FaDu resistant by microarray of expression. DAPI (Hoescht) staining in blue. p-EGFR-FITC+ p-mTOR-FITC+. Arrows indicate EGFR-FITC+ and p-mTOR-FITC+ localization. The images were acquired in 40× magnification. The number under the bands represented relative ratios (phospho/total).

Figure 6

Malignant phenotype acquired after cetuximab resistance establishment. (A) Representative images of wound healing assay of FaDu parental and FaDu resistant cell lines in 24, 48, and 72 h. The yellow lines represent the distance between both edges of the wound; Scale bars, 200 µm; (B) Migration rates of FaDu parental and FaDu resistant cells in a wound-healing assay; (C) Representative images of adhesion and clonogenic assay for parental and resistant cells; (D) The absolute number of adherent cells; (E) The absolute number of colonies in clonogenic cell assay for anchorage-dependent in parental and resistant cells. (*** p < 0.0001). The images were acquired in 10× magnification.

Figure 7

Epithelial–mesenchymal transition (EMT) markers expression in FaDu parental and FaDu resistant cells. (A) Representative images of EMT proteins detected in Western blot assay in parental and resistant cells. (B) E-cadherin densitometry. (C) N-cadherin densitometry. (D) α-smooth densitometry. (E) Slug densitometry. (F) Snail densitometry (G) TGF-β densitometry (H) CD44 densitometry Data are presented in fold-change in comparison with FaDu parental. Fadu p: FaDu parental; FaDu R: FaDu resistant. (*** p < 0.001).