Bosutinib Inhibits EGFR Activation in Head and Neck Cancer

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and although new therapeutic approaches have been recently evaluated, overall patient survival is still poor. Thus, new effective and selective clinical treatments are urgently needed. An analysis of data from large-scale, high-throughput drug screening cell line projects identified Bosutinib, a Src/Abl inhibitor that is currently used for the treatment of chronic myelogenous leukemia, as a candidate drug to treat HNSCC. Using a panel of HNSCC-derived cell lines, we found that treatment with Bosutinib reduced cell proliferation and induced apoptosis of sensitive cell lines. The drug rapidly inhibited Src and EGFR (epidermal growth factor receptor) phosphorylation, and sensitivity to Bosutinib was correlated with the activation status of EGFR. Similar findings were observed in in vivo xenograft assays using HNSCC derived cells. Moreover, in the presence of mutations in PIK3CA, the combination of Bosutinib with the PI3Kα inhibitor Alpelisib showed a synergistic effect. These results suggest that Bosutinib could be a new effective drug for the treatment of HNSCC, particularly in tumors with high EGFR activity. Its combination with Alpelisib could especially benefit patients bearing activating mutations of PIK3CA.

Keywords: Alpelisib; Bosutinib; EGFR inhibitors; cancer cell lines; head and neck cancer; targeted therapies.

Figure 1

Sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to Bosutinib. (A) Cell viability as measured by XTT. Data represent means ± SEMs from a representative experiment of at least three different experiments for each cell line (each concentration point was replicated six times within each experiment). The cell viability curves (continuous, broken or dotted) for each cell line are a non-linear regression fit of the data (log inhibitor versus normalized response). (B) Cell cycle analysis of Bosutinib-treated HNSCC cell lines. Cells were treated with their corresponding IC₇₅ for 24 h and the cell cycle was analyzed by flow cytometry. The graphs represent the percentage of cells in each phase of the cell cycle. Data represent means ± SEMs from at least two different experiments (each point was replicated three times within the experiment). * Cell lines with mutations in PIK3CA. In red are cell lines resistant to Bosutinib; in blue are cell lines sensitive to Bosutinib.

Figure 2

(A) Src and epidermal growth factor receptor (EGFR) phosphorylation was analyzed by Western blot analysis. The numbers indicate the normalized ratio of the phosphorylated form versus the total form. (B) Linear regression of the p-EGFR/EGFR ratio versus sensitivity to Bosutinib (IC₅₀) of the different HNSCC cell lines. EGFR and PIK3CA copy number (C) and mRNA expression (D). * Cell lines with mutations in PIK3CA. In red are cell lines resistant to Bosutinib; in blue are cell lines sensitive to Bosutinib.

Figure 3

Western blot analysis of Src and EGFR phosphorylation of Bosutinib-sensitive HNSCC cell lines treated with the IC₅₀ of the drug at different time points. * Cell lines with mutations in PIK3CA.

Figure 4

Effect of Bosutunib in HNSCC xenografts. (A) Cal33-derived xenografts were treated as indicated. P.o.: Per Os (oral administration); i.p.: intraperitoneal. (B) Western blot with the indicated antibodies of lysates from tumors treated as specified for 10 days. Each lane is a lysate from a tumor corresponding to the indicated treatment group. (C) Tumor growth of Cal33-derived xenografts treated as indicated (n = 10 per arm). * p < 0.05, ** p < 0.01. (D) Sections from tumors treated as indicated for 10 days were immunostained with BrdU to assess proliferation, the epithelial marker keratin 5 (K5), the pan keratin marker AE1/AE, and the apoptotic marker Caspase 3. Scale bar 100 µm.

Figure 5

(A) Western blot analysis of ERK, Akt and S6 phosphorylation of Cal33 cells treated with the IC₅₀ of Bosutinib at different time points. (B) Western blot analysis of ERK, Akt and S6 phosphorylation of Cal33 cells treated with Bosutinib (IC₅₀), Alpelisib (IC₅₀) and Erlotinib (10 µM) at 6 h. * Mutated in PIK3CA. Black arrows indicate p-Akt Thr308.

Figure 6

Sensitivity of HNSCC cell lines to Bosutinib in combination with Alpelisib. (A) Cell viability, as measured by XTT of the indicated cell lines treated with Bosutinib, Alpelisib and their combination at the indicated amounts of their IC₅₀ dose. Data represents means ± SEMs (each concentration point was replicated four times within the experiment). (B) Fa–CI plots to measure the synergism of the combination of two drugs. Fa represents the fraction of the cells affected by the combination of both drugs (at the concentrations indicated in panel A), “0” being 100% cell survival and “1” being 0% cell survival. The Combination Index (CI) was calculated using Compusyn software and plotted for each combination. (C) Description of the CI values. * Cell lines with mutations in PIK3CA. In red are cell lines resistant to Bosutinib; in blue are cell lines sensitive to Bosutinib.