Evaluation of co‑inhibition of ErbB family kinases and PI3K for HPV‑negative head and neck squamous cell carcinoma

Abstract

The ErbB/HER family of protein‑tyrosine kinases and PI3K represent crucial targets in the treatment of head and neck squamous cell carcinoma (HNSCC). A combination therapy of afatinib (ErbB inhibitor) and copanlisib (PI3K inhibitor), both Food and Drug Administration‑approved kinase inhibitors, can suppress the growth of human papillomavirus (HPV)‑positive HNSCC. The current study further evaluated the efficacy and clinical potential of this combination therapy for the treatment of HPV‑negative HNSCC in vitro and in vivo. Sulforhodamine B cell viability assay and Annexin V/propidium iodide staining demonstrated that this combination treatment markedly enhanced inhibition of cell viability and reduced cell survival when compared with treatment with either inhibitor alone in two HPV‑negative HNSCC cell lines. Notably, this combination also led to significant inhibition of xenograft tumor growth in mice, without any apparent effects on body weight. Western blot analysis found that copanlisib alone effectively blocked PI3K/Akt signaling but caused upregulation of HER2 and HER3 phosphorylation, as reported in other types of cancer. However, the combination of copanlisib and afatinib completely blocked phosphorylation of the ErbB family (including HER3) and Akt, while also increasing apoptosis. In conclusion, these results suggested that co‑targeting the ErbB family kinases and PI3K using a combination treatment of afatinib and copanlisib may have clinical potential for patients with HPV‑negative HNSCC.

Keywords: ErbB inhibitor; PI3K inhibitors; head and neck squamous cell carcinoma; targeted therapies.

Figure 1.

Afatinib more effectively inhibits the viability of human papillomavirus-negative head and neck squamous cell carcinoma cells compared with gefitinib and erlotinib. (A) Cal27 and (B) FaDu cells were treated with DMSO or increasing concentrations of gefitinib (3 nM-30 µM), erlotinib (3 nM-30 µM) and afatinib (1 nM-10 µM) at 37°C for 96 h. Cell viability was measured using the sulforhodamine B cell viability assay and EC₅₀ values were determined using GraphPad Prism version 10.3.1. The experiments were performed in triplicate and repeated at least three times. EC₅₀, half maximal effective concentration.

Figure 2.

Copanlisib more effectively inhibits cell viability compared with other PI3K inhibitors in Cal27 cells. Cal27 cells were treated with DMSO or increasing concentrations of copanlisib (0.01 nM-10 µM) and other PI3K inhibitors (0.01 nM-100 µM) at 37°C for 96 h. Cell viability was measured using the sulforhodamine B assay and the viability curves are shown. The experiments were performed in triplicate and repeated at least three times. The associated EC₅₀ values of the inhibitors were determined using GraphPad Prism version 10.3.1.

Figure 3.

Synergistic inhibition of cell viability by the combination of afatinib and copanlisib in vitro. (A) Cal27 and (B) FaDu cells were treated with two-fold diluted concentrations of afatinib, copanlisib or their combination at 37°C for 96 h with the half maximal effective concentration values in the middle. Cell viability was measured using the sulforhodamine B cell viability assay. The experiments were performed in triplicate and repeated twice due to the consistent results each time. (C) CI values of the combination of various concentrations of copanlisib (2.875–92 nM) and afatinib (0.2875–9.2 nM) in Cal27 cells were determined using CalcuSyn version 2.0. (D) CI values of the combination of various concentrations of copanlisib (3.375–118 nM) and afatinib (0.3125–11.6 nM) in FaDu cells were determined using CalcuSyn version 2.0. CI, combination index.

Figure 4.

Inhibition of head and neck squamous cell carcinoma growth by a combination of afatinib and copanlisib in vivo. FaDu cells were inoculated into mice. When tumors reached ~150 mm³, mice were randomized to one of four treatment groups (n=6/group): Vehicle control, copanlisib (6 mg/kg, intraperitoneal, 5 times/week Monday to Friday), afatinib (6 mg/kg, oral, 5 times/week, Monday to Friday), or a combination of copanlisib and afatinib. The treatments were performed for 32 days. (A) Xenograft tumor volumes and images, (B) final average weights of tumors, and (C) average body weight changes of mice in each group were compared by GraphPad Prism 10.3.1 software. After one-way ANOVA, Tukey's post hoc test was performed for multiple comparisons. ***P<0.001; **P<0.01; *P<0.05.

Figure 5.

A combination of afatinib and copanlisib increases cell apoptosis compared with afatinib or copanlisib treatment alone. (A) Cal27 and (B) FaDu cells were treated with vehicle control, copanlisib, afatinib or a combination for 48 h, and cell apoptosis was analyzed by Annexin V/propidium iodide staining. The experiments were performed in triplicate and repeated at least three times. Early and late-stage apoptotic, and dead cells were counted, and statistical analysis was performed by GraphPad Prism 10.3.1 software. After ANOVA, Tukey's post hoc test was performed for multiple comparisons. ****P<0.0001; ***P<0.001; **P<0.01.

Figure 6.

A combination of copanlisib and afatinib effectively blocked the phosphorylation of HER2, HER3 and Akt, and enhanced caspase-3 cleavage. (A) Cal27 and (B) FaDu cells were treated with increasing concentrations of copanlisib, 0.5 µM afatinib or their combination for 24 h before lysis. The indicated proteins were detected by western blot analysis. C-, cleaved; P-, phosphorylated.