Differential modulation of PI3K/Akt/mTOR activity by EGFR inhibitors: A rationale for co-targeting EGFR and PI3K in cisplatin-resistant HNSCC

Abstract

Purpose: To find a new strategy to treat cisplatin-resistant head and neck squamous cell carcinoma (HNSCC), we investigated the effects of EGFR inhibitors on the PI3K/Akt/mTOR pathway and determined the efficacy of EGFR inhibitors in combination with PI3K inhibitors to suppress cell proliferation in cisplatin-resistant-HNSCC.

Methods: The cisplatin-resistant HNSCC cell lines were treated with four FDA approved EGFR inhibitors, which included Gefitinb or Erlotinib alone, or in combination with the pan-PI3K inhibitor, BKM120. Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell proliferation was examined by MTS assay. Apoptosis was analyzed by flow cytometry.

Results: Cisplatin-resistant HNSCC cells were also resistant to EGFR inhibitors. However, a combination of EGFR inhibitors with PI3K inhibitor BKM120 dramatically improved the efficacy of EGFR inhibitors to inhibit cell proliferation and induce apoptosis. Furthermore, treatment with EGFR inhibitors differentially affected the phosphorylation of Akt and mTOR, which included partial inhibition, no inhibition, and induction. A combination of EGFR inhibitors and BKM120 completely blocked phosphorylation of EGFR, Akt, and S6K (an mTOR target).

Conclusion: Our data provided a rationale for EGFR inhibitors in combination with PI3K inhibitors to treat cisplatin-resistant HNSCC.

Keywords: EGFR; EGFR inhibitor; HNSCC; PI3K; PI3K inhibitors; cisplatin resistance; targeted therapy.

Figure 1.. Synergistic inhibition of cell proliferation by combination of Gefitinib and BKM120.

A and B. Cal27CP (A) and SCC25CP (B) cells were treated with Gefitinib (10 μM), increasing concentrations of BKM120, or their combinations for 72 hours and cell proliferation was measured by MTS assay. The experiments were performed in triplicate, and the results are representative of three independent experiments. The combination index values (CI values) for different combinations were determined using CalcuSyn software. C and D. Cal27CP (C) and SCC25CP (D) cells were treated with DMSO, Gefitinib, BKM120, or their combinations for 10 days and colony formation was observed. Each experiment was performed in triplicate.

Figure 2.. Synergistic inhibition of cell proliferation by combination of Erlotinib and BKM120.

A and B. Cal27CP (A) and SCC25CP (B) cells were treated with Erlotinib (10 μM), increasing concentrations of BKM120, or their combinations for 72 hours and cell proliferation was measured by MTS assay. The experiments were performed in triplicate, and the results are representative of three independent experiments. The combination index values (CI values) for different combinations were determined using CalcuSyn software. C and D. Cal27CP (C) and SCC25CP (D) cells were treated with DMSO, Erlotinib, BKM120, or their combinations for 10 days and colony formation was observed. Each experiment was performed in triplicate.

Figure 3.. Gefitinib and BKM120 cooperated to induced apoptosis.

A. Cal27CP cells were treated with vehicle control, Gefitinib (10 μM), BKM120 (1 μM), or their combinations for 48 hours and cell apoptosis was measured by Annexin V. The experiments were performed in triplicate, early and late-stage apoptosis and dead cells were counted, and statistical analysis was performed. P values < 0.05 were statistically significant (***P=0.0007; ****P<0.0001). B. SCC25CP cells were treated with vehicle control, Gefitinib (10 μM), BKM120 (1 μM), or their combinations for 48 hours and cell apoptosis was measured by Annexin V. The experiments were performed in triplicate, early and late-stage apoptosis and dead cells were counted, and statistical analysis was performed. P values < 0.05 were statistically significant (***P=0.001; ****P<0.0001).

Figure 4.. Induction of apoptosis by a combination of Erlotinib and BKM120.

A. Cal27CP cells were treated with vehicle control, Erlotinib (10 μM), BKM120 (1 μM), or their combinations for 48 hours and cell apoptosis was measured by Annexin V. The experiments were performed in triplicate, early and late-stage apoptosis and dead cells were counted, and statistical analysis was performed. P values < 0.05 were statistically significant (***P=0.0005; ****P<0.0001). B. SCC25CP cells were treated with vehicle control, Erlotinib (10 μM), BKM120 (1 μM), or their combinations for 48 hours and cell apoptosis was measured by Annexin V. The experiments were performed in triplicate, early and late-stage apoptosis and dead cells were counted, and statistical analysis was performed. P values < 0.05 were statistically significant (**P=0.0018; ****P<0.0001).

Figure 5.. Gefitinib differentially affected phosphorylation of Akt and S6K, whereas the combination of Gefitinib and BKM120 significantly blocked phosphorylation of Akt and S6K in Cisplatin-resistant HNSCC cells.

A-C. Cal27CP (A), SCC25CP (B), and UMSCC74B (C) cells were treated with increasing concentrations of Gefitinib, BKM120 (2.5 μM), or their combination for 24 hours, cells were lysed, and the indicated proteins were detected by Western blot analysis. Note: the arrows indicated the bands of P-S6K or total S6K.

Figure 6.. Erlotinib differentially modulated phosphorylation of Akt and S6K, whereas the combination of Gefitinib and BKM120 significantly blocked phosphorylation of Akt and S6K in Cisplatin-resistant HNSCC cells.

A-C. Cal27CP (A), SCC25CP (B), and UMSCC74B (C) cells were treated with increasing concentrations of Erlotinib, BKM120 (2.5 μM), or their combination for 24 hours, cells were lysed, and the indicated proteins were detected by Western blot analysis. Note: the arrows indicated the bands of P-S6K or total S6K.