Simultaneous targeting of EGFR, HER2, and HER4 by afatinib overcomes intrinsic and acquired cetuximab resistance in head and neck squamous cell carcinoma cell lines

Abstract

The epidermal growth factor receptor (EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR-targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti-EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first-generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines, independent of the HPV status. However, cross-resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G₀ /G₁ cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.

Keywords: afatinib; cetuximab; epidermal growth factor receptor; head and neck squamous cell carcinoma; human papillomavirus; resistance.

Figure 1

Dose–response curves of cetuximab (168 h) evaluated using the SRB assay. (A) Dose–response curves for HPV‐positive and HPV‐negative HNSCC cell lines. Five of seven HNSCC cell lines (i.e., LICR‐HN1, Cal‐27, SQD9, 93‐VU‐147T, and UM‐SCC104) were considered as intrinsically resistant to cetuximab versus SC263 and SCC22b, which were cetuximab sensitive. (B) Dose–response curves for isogenic cetuximab‐resistant (SCC22b‐R) and cetuximab‐sensitive (SCC22b‐PBS) HNSCC cell lines. (C) Dose–response curve for the cetuximab‐resistant cell line SCC22b‐R after 6 weeks of culture in drug‐free medium, followed by cetuximab treatment for 168 h. This graph represents one experiment executed in threefold.

Figure 2

Protein levels of HER receptors under normoxic and hypoxic conditions in a panel of HNSCC cell lines with different sensitivity to cetuximab. The percentage of EGFR‐, HER2‐, and HER3‐positive cells (overton) are presented in A, C, and E, respectively. The expression levels of EGFR, HER2, and HER3 on the corresponding receptor‐positive cells (ΔMFI) are presented in B, D, and F, respectively. Protein levels were measured with the FACScan flow cytometer.

Figure 3

Effect sizes with standard errors for the influence of hypoxia on the expression of HER family members in HNSCC cell lines with different cetuximab resistance status. The effect size represents the difference in mean overton (A) and ΔMFI (B) between normoxia and hypoxia. Positive and negative effect sizes, respectively, indicate an increase and decrease under hypoxia compared to normoxia. The * indicates a significant P‐value for the main effect of oxygen condition.

Figure 4

Afatinib's cytotoxicity in cetuximab‐sensitive and cetuximab‐resistant HNSCC cell lines. (A) Dose–response curves of HPV‐positive and HPV‐negative intrinsically cetuximab‐resistant HNSCC cell lines after exposure to afatinib for 72 h under normoxia. (B) Dose–response curves for the cetuximab‐sensitive HNSCC cell lines after exposure to afatinib for 72 h under normoxia. (C) Dose–response curves of acquired cetuximab‐resistant (suffix R) and corresponding cetuximab‐sensitive isogenic cell lines (suffix PBS) after exposure to afatinib for 72 h under normoxia. (D) Effect sizes with standard errors for the influence of hypoxia on afatinib's cytotoxic effect in HNSCC cell lines with different cetuximab resistance status. The average log(IC ₅₀) of afatinib was higher under normoxia compared to hypoxia in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. The effect size represents the difference in mean log(IC ₅₀) between normoxia and hypoxia. The * indicates a significant P‐value for the main effect of oxygen condition.

Figure 5

Cell cycle distribution in HNSCC cell lines after afatinib treatment (0 nm, IC ₂₀, IC ₄₀, IC ₆₀) under both normoxic and hypoxic conditions. Cells were stained with PI and DNA content was measured by flow cytometric analysis. Cells were divided into 3 groups: G₀/G₁ phase (2n), S phase (2n–4n), and G₂/M phase (4n). Treatment with afatinib led to an increase of the proportion of cells in the G₀/G₁ phase of the cell cycle in the majority of HPV‐negative (A–C) and HPV‐positive (D, E) intrinsically cetuximab‐resistant HNSCC cell lines under both normoxia and hypoxia. This G₀/G₁ cell cycle arrest was also observed in cetuximab‐sensitive (F, I) as well as isogenic acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines (G, H, J, and K). *P‐value effect treatment on the percentage G₀/G₁ cells ≤ 0.050.

Figure 6

Induction of apoptotic cell death in HNSCC cell lines after afatinib treatment (0 nm, IC ₂₀, IC ₄₀, IC ₆₀, IC ₈₀) under both normoxic and hypoxic conditions. Cells were stained with annexin V‐FITC (AnnV) and PI and measured flow cytometrically. Treatment with afatinib induced an increase in the percentage of AnnV+/PI− and AnnV+/PI+ cells with a corresponding decrease of the percentage viable (AnnV−/PI−) cells in the majority of HPV‐negative (A–C) and HPV‐positive (D, E) intrinsically cetuximab‐resistant HNSCC cell lines under both normal and reduced oxygen conditions. This induction of apoptotic cell death was also observed in cetuximab‐sensitive (F, I) as well as acquired cetuximab‐resistant and PBS‐treated control (G, H, J, and K) HNSCC cell lines. *P‐value effect treatment on the percentage AnnV+/PI− cells ≤ 0.050.

Figure 7

Caspase‐3/7 activity in HNSCC cell lines during afatinib treatment (0 nm, IC ₄₀, IC ₈₀) under normoxic conditions. The induction of apoptosis was detected by real‐time measurements of active caspase‐3/7 during afatinib treatment using the IncuCyte system. The green object count corresponds with the caspase‐3/7 activity. After 24 h, treatment with afatinib induced a significant increase in caspase‐3/7 activity in the majority of HPV‐negative (A–C) and HPV‐positive (D, E) intrinsically cetuximab‐resistant HNSCC cell lines. This significant increase in caspase‐3/7 activity was also detected in cetuximab‐sensitive (F, I), acquired cetuximab‐resistant, and PBS‐treated control (G, H, J, and K) HNSCC cell lines. *P‐value effect treatment on the green object count ≤ 0.050.

Figure 8

The cytotoxic effects of afatinib followed by cisplatin treatment in a panel of HNSCC cell lines with different sensitivity to cetuximab. Dose–response curves for the intrinsically cetuximab‐resistant cell lines LICR‐HN1 (A), SQD9 (B), and Cal‐27 (C) indicate an additive to antagonistic effect. Dose–response curves for the cetuximab‐sensitive cell lines SC263 (D) and SCC22b (E) show an additive to subadditive effect. Survival curves were corrected for the cytotoxic effect of 72‐h afatinib alone. Cells were treated with fixed concentrations afatinib, which were based on the outcome of the monotherapy experiments.

Figure 9

The cytotoxic effect of cisplatin followed by afatinib treatment in a panel of HNSCC cell lines with different sensitivity to cetuximab. Dose–response curves for the intrinsically cetuximab‐resistant cell lines LICR‐HN1 (A), SQD9 (B), and Cal‐27 (C) also indicate an additive to antagonistic effect. Dose–response curves for the cetuximab‐sensitive cell lines SC263 (D) and SCC22b (E) show an additive to antagonistic effect. Survival curves were corrected for the cytotoxic effect of 72‐h afatinib alone. Cells were treated with fixed concentrations afatinib, which were based on the outcome of the monotherapy experiments.