Inhibition of cisplatin-resistant head and neck squamous cell carcinoma by combination of Afatinib with PD0325901, a MEK inhibitor

Abstract

ErbB family members that contain EGFR, HER2, HER3 and HER4 play important roles in many cancer types, including head and neck; however, inhibition of these receptors by small molecule kinase inhibitors showed limited results due to compensatory up-regulation of some key survival signaling pathways. Here, we explore the effectiveness of Afatinib, an irreversible inhibitor of EGFR, HER2, and HER4, in combination with the MEK inhibitor PD0325901 to inhibit cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We treated two cisplatin-resistant HNSCC cell lines, UMSCC74B and O28, with Afatinib, PD0325901, or a combination, and measured signaling pathways, cell proliferation, and survival. We found that Afatinib blocked Akt/mTOR activity and phosphorylation of EGFR, HER2 and HER3, but up-regulated MEK/ERK signaling. Interestingly, MEK inhibitor PD0325901 blocked ERK phosphorylation, but elevated phosphorylation of Akt and mTOR pathways. Similarly, Afatinib and PD0325901 inhibited all these pathways and synergistically suppressed cell proliferation and survival. Our data demonstrate that Afatinib in combination with MEK inhibitors could provide a potential novel therapy for cisplatin-resistant head and neck squamous cell cancer.

Keywords: Afatinib; Akt; EGFR; EGFR inhibitors; ERK; HNSCC; Head and neck squamous cell carcinoma; MEK; MEK inhibitor; PD0325901; PI3K; cisplatin-resistant HNSCC; mTOR.

Figure 1

Afatinib inhibits EGFR, HER2, and HER3, as well as Akt/mTOR signaling, but induces MEK/ERK pathway in cisplatin-resistant HNSCC cells. A: Cell lysates were prepared from UMSCC74B (Left) and O28 (Right) treated with different doses of Afatinib for 24 hours and phosphorylation and total levels of EGFR, HER2, HER3, Akt, S6, and ERK, as well as GAPDH expression were detected by Western blot analysis. B: Inhibition of EGFR, HER2, HER3, Akt, S6, and ERK, but induction of ERK pathway by Afatinib.

Figure 2

MEK inhibitor PD0325901 inhibits ERK, but induces Akt/mTOR signaling pathway in cisplatin-resistant HNSCC cells. A: Cell lysates were prepared from UMSCC74B (Left) and O28 (Right) treated with different doses of PD0325901 for 24 hours and phosphorylation and total levels of ERK, Akt, and S6, as well as GAPDH expression were detected by Western blot analysis. B: Inhibition of ERK, but induction of Akt/mTOR pathways by PD0325901.

Figure 3

Inhibition of ErbB family, Akt/mTOR, and MEK/ERK by a combination of Afatinib and PD0325901 in cisplatin-resistant HNSCC. A: Cell lysates were prepared from UMSCC74B cells treated with different doses of PD0325901 for 48 (Left) or 72 (Right) hours and phosphorylation and total levels of Akt and ERK, as well as GAPDH expression were detected by Western blot analysis. B: Cell lysates were prepared from O28 cells treated with different doses of PD0325901 for 48 (Left) or 72 (Right) hours and phosphorylation and total levels of Akt and ERK, as well as GAPDH expression were detected by Western blot analysis. C: Inhibition of Akt and ERK following combination treatment with Afatinib and PD0325901.

Figure 4

Afatinib and PD0325901 synergistically induces apoptosis in UMSCC74B cells. (A) Cleaved caspase-3 in cell lysates from Figure 3A was detected by Western blot. (B) Cells were treated with vehicle control, Afatinib, PD0325901, or a combination for 48 hours. Cell apoptosis was measured by Annexin V. (C) Experiments in (B) were performed in triplicate, early and late stage apoptosis counted, and statistical analysis performed. P values less than 0.05 were considered to be statistically significant.

Figure 5

Afatinib and PD0325901 cooperate to induce apoptosis in O28 cells. (A) O28 cells were treated with vehicle control, Afatinib, PD0325901, or a combination for 48 hours before cells were lysed; cleaved caspase-3 in cell lysates was detected by Western blot. (B) Cells were treated with vehicle control, Afatinib, PD0325901, or a combination for 48 hours. Cell apoptosis and death were measured by Annexin V. (C) Experiments in (B) were performed in triplicate, early and late stage apoptosis counted, and statistical analysis performed. P values less than 0.05 were considered to be statistically significant.

Figure 6

Afatinib and PD0325901 synergistically cooperate to suppress UMSCC74B cell proliferation. A: Synergistic suppression of cell growth by Afatinib and PD0325901. UMSCC74B cells were treated with vehicle control, Afatinib, PD0325901, or a combination for 48 hours and cell density was observed microscopically. B: Afatinib and PD0325901 synergistically inhibit cell proliferation. UMSCC74B cells were treated with DMSO or different doses of Afatinib, PD0325901, or a combination for 72 hours and cell proliferation was determined by MTS assay. The experiments were performed in triplicate, and the combination index values (CI values) were determined using CalcuSyn software.

Figure 7

Afatinib and PD0325901 cooperate to suppress O28 cell proliferation. A: O28 cells were treated with vehicle control, Afatinib, PD0325901 or a combination for 72 hours and cell density was observed microscopically. B: O28 cells were treated with DMSO or different doses of Afatinib, PD0325901, or a combination for 72 hours and cell proliferation was determined by MTS assay. The experiments were conducted twice in triplicate and the combination index values (CI values) were determined using CalcuSyn software.