IKK phosphorylation of NF-κB at serine 536 contributes to acquired cisplatin resistance in head and neck squamous cell cancer

Abstract

Current treatment methods for advanced head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy and chemotherapy. For recurrent and metastatic HNSCC, cisplatin is the most common treatment option, but most of patients will eventually develop cisplatin resistance. Therefore, it is imperative to define the mechanisms involved in cisplatin resistance and find novel therapeutic strategies to overcome this deadly disease. In order to determine the role of nuclear factor-kappa B (NF-κB) in contributing to acquired cisplatin resistance in HNSCC, the expression and activity of NF-κB and its upstream kinases, IKKα and IKKβ, were evaluated and compared in three pairs of cisplatin sensitive and resistant HNSCC cell lines, including a pair of patient derived HNSCC cell line. The experiments revealed that NF-κB p65 activity was elevated in cisplatin resistant HNSCC cells compared to that in their parent cells. Importantly, the phosphorylation of NF-κB p65 at serine 536 and the phosphorylation of IKKα and IKKβ at their activation loops were dramatically elevated in the resistant cell lines. Furthermore, knockdown of NF-κB or overexpression of p65-S536 alanine (p65-S536A) mutant sensitizes resistant cells to cisplatin. Additionally, the novel IKKβ inhibitor CmpdA has been shown to consistently block the phosphorylation of NF-κB at serine 536 while also dramatically improving the efficacy of cisplatin in inhibition of cell proliferation and induction of apoptosis in the cisplatin resistant cancer cells. These results indicated that IKK/NF-κB plays a pivotal role in controlling acquired cisplatin resistance and that targeting the IKK/NF-κB signaling pathway may provide a possible therapeutic method to overcome the acquired resistance to cisplatin in HNSCC.

Keywords: HNSCC; IKK; IKKβ inhibitors; NF-κB; cisplatin resistance.

Figure 1

Characterization of cisplatin resistance in head and neck cancer cell lines. A. IC₅₀ determination of cisplatin in UMSCC-17B, UMSCC-17B/CP, UMSCC-25 and UMSCC-25/CP. Cells were treated with cisplatin (0-100 μM) for 72 hours at 37°C. Representative results from at least three experiments for each of the two treatments are shown. Error bars represent standard error of mean of three wells (*, P < 0.05). B. Cisplatin induced caspase 3/7 activity in parental but not resistant cells. Cells were treated with cisplatin (20 μM) for 48 hours. Caspase 3/7 activity was evaluated following the protocol. The results are representative of three independent experiments (**, P < 0.01). C. Cisplatin induced caspase-3 cleavage in parental but not resistant cells. The indicated cells were treated with cisplatin (0-20 μM) for 24 hours and lysed and cleaved caspase 3 was measured by western blot. The results are representative of three independent experiments.

Figure 2

NF-κB/IKK signaling is upregulated in cisplatin resistant HNSCC.A. IKK/NF-κB activity is elevated in the cisplatin resistant cell lines. The levels of phosphorylation of IKKα/β and p65, IKKα, IKKβ and GAPDH in the cells were determined by western blot. Densitometric analysis of 3 independent experiments shows a statistically-significant increase in levels of phosphorylated p65 in both cell lines. B. Cells were transfected with siRNA control, IKKα, IKKβ or IKKα plus IKKβ and blotted with the indicated antibodies. C. NF-κB p65 translocate to nucleus in cisplatin resistant cell lines. The levels of p65, β-tublin and Lamin A/C of the cytoplasmic and nuclear extracts of the cells were determined by immunoblotting with the indicated antibodies. Densitometry analyses for p65 were performed employing ImageJ software (NIH) and presented as ratio of nuclear p65 to cytoplasmic p65 band signal intensity. D. NF-κB reporter activity increased in cisplatin resistant cells. Cells were harvested 24 hours after transfection with luciferase reporter and Renilla reporter control and luciferase assays were performed. The experiments were repeated three times (**, P < 0.01).

Figure 3

Depletion of NF-κB sensitizes cisplatin resistant cells to cisplatin treatment. A. The siRNA against p65 is effective in reducing p65 expression in the cisplatin resistant cells. Cells were transfected with siRNA control or siRNA NF-κB p65 for 48 hours and lysed and the levels of the levels of p65 and GAPDH were determined by immunoblotting. The experiments were carried out on three separate occasions. B. Cells were transfected with siRNA control or siRNA NF-κB p65 for 48 hours and then treated (DMSO) or Cisplatin (20 μM) for additional 24 hours. Caspase 3/7 activity was evaluated following the protocol. The results are representative of three independent experiments (*, P < 0.05, **, P < 0.01). C. Cells were transfected with siRNA control or siRNA NF-κB p65 for 48 hours and then left untreated (DMSO) or treated with 20 μM Cisplatin for additional 48 hours. The levels of cleaved caspase 3, p65 and GAPDH were determined by immunoblotting. The experiments were repeated three times.

Figure 4

Overexpression of NF-κB serine 536 alanine mutant sensitizes cisplatin resistant cells to cisplatin. A. Cells were transfected with vector or flag-p65 S536A and left untreated or treated with cisplatin for 48 hours and caspase cleavage was measured by western blot. B. Cells were transfected with vector or flag-p65 S536A and left untreated or treated with cisplatin for 48 hours and caspase activity was measured (*, P<0.05).

Figure 5

Inhibition of NF-κB/IKKβ signaling by the IKKβ inhibitor, CompA sensitizes cisplatin resistant cells to cisplatin. A. Cells were treated with different doses of CompA for 2 hours, lysed and analyzed by western blots. B. Cells were treated with CompA alone, cisplatin alone, or CompA and cisplatin together for 48 hours and Caspase 3/7 activity was evaluated (*, P < 0.05; **, P < 0.01). C. Cells were treated with CompA alone, cisplatin alone, or CompA and cisplatin together for 48 hours and cleaved caspase 3 and GAPDH were determined. The experiments were repeated three times. D. Cells were pre-treated with CompA, cisplatin, or CompA plus cisplatin and colony formation was observed and the numbers of colony were counted. The results are representative of three independent experiments.

Figure 6

NF-κB is activated in patient-derived HNSCC cell lines and cisplatin resistance is reversed with IKKβ inhibition. A. IC₅₀ values of UMSCC-11A and UMSCC-11B cells were determined. Cells were treated with Cisplatin (0-100 μM) for 72 hours. Cell proliferation was measured through MTT Assay. IC₅₀ values were determined. B. Cell lysates from UMSCC-11A and UMSCC-11B cells were analyzed by western blots. C. Cells were treated with different doses of CompA for 2 hours, lysed and analyzed by western blots. D. UMSCC-11B cells were treated with CompA alone, Cisplatin alone, or CompA and Cisplatin as indicated for 48 hours and cell proliferation was measured by MTT assay. The results are representative of three independent experiments (*, P < 0.05). E. Cells were treated same with (C) for 48 hours and Caspase 3/7 activity was measured. The results are representative of three independent experiments (*, P < 0.05). F. Cells were treated same with (C) and the levels of cleaved caspase 3 and GAPDH. The results are representative of three independent experiments. G. Cells were treated with CompA, Cisplatin, or CompA and Cisplatin as indicated and colony formation was observed and the numbers of colony were counted. Each experiment was repeated three times.