Inhibition of IKKβ/NF-κB signaling pathway to improve Dasatinib efficacy in suppression of cisplatin-resistant head and neck squamous cell carcinoma

Abstract

Proto-oncogene tyrosine-protein kinase Src plays an important role in Head and Neck Squamous Cell Carcinoma (HNSCC). However, the FDA-approved SRC inhibitor Dasatinib shows very limited efficacy in HNSCC clinical trials, even though Dasatinib can completely inhibit SRC in the laboratory setting. These results suggest that SRC inhibition can cause compensatory up-regulation and/or activation of other survival pathways, which suggests that co-targeting of SRC and the potential signaling pathways may improve the Dasatinib efficacy. In this study, we investigated the role of IKKβ/NF-κB in regulation of the sensitivity of cisplatin-resistant HNSCC to Dasatinib. Additionally, we wished to determine whether inhibition of the IKKβ/NF-κB signaling pathway could enhance Dasatinib efficacy to inhibit cisplatin-resistant HNSCC without the use of cisplatin. Previous studies have shown that ETS-1 is a crucial SRC effector protein that regulates cancer cell proliferation, anti-apoptosis, and metastasis. We found that SRC kinase inhibition by Dasatinib decreased ETS-1 expression but caused elevation of IKKβ/NF-κB signaling in multiple cisplatin-resistant HNSCC. Interestingly, inhibition of IKKβ/NF-κB by CmpdA (Bay65-1942), a recently identified IKKβ inhibitor, also led to a decrease in ETS-1 levels. Moreover, the knockdown of IKK, but not NF-κB, dramatically decreased ETS-1 expression. In addition, IKKβ and ETS-1 interacted in cisplatin-resistant HNSCC. These data demonstrated cross-talk between SRC and IKK to regulate NF-κB and ETS-1. Furthermore, we found that simultaneous inhibition of SRC and IKKβ through a Dasatinib and CmpdA combination synergistically inhibited NF-κB activation and ETS-1expression, suppressed cell proliferation, and induced apoptosis. Taken together, our data indicate that SRC and IKKβ play crucial roles in cisplatin-resistant HNSCCC and co-targeting SRC and IKKβ could be an effective strategy to treat cisplatin-resistant HNSCC.

Keywords: Targeted therapies; Translational research.

Fig. 1. Dasatinib inhibits SRC and ETS-1 but induces IKKβ/NF-κB in cisplatin resistant HNSCC.

a–c Cell lysates were prepared from Cal27CP (a), SCC25C (b), and FaDu-CP (c) cells treated with Dasatinib for 8 h and phosphorylation and total levels of SRC, p65, IKK, and expression of ETS-1 and β-actin were detected by Western blot analysis. The density of P-p65, p65, and β-actin bands was quantitated and the level of phosphorylation of p65 was normalized by p65 and β-actin. d Cal27CP cells were treated with DMSO or 200 nM Dasatinib for 24 h, mRNA was extracted and the expression of IL-6 was detected by real time PCR.

Fig. 2. Inhibition of IKKβ decreased ETS-1 expression.

Cal27CP (a), SCC25C (b), and FaDu-CP (c) cells were treated with increasing concentrations of IKKβ inhibitor, CmpdA, for 24 h and phosphorylation and total levels of SRC, p65, and expression of ETS-1 and β-actin were detected by Western blot analysis.

Fig. 3. IKK regulates ETS-1 independent of NF-κB.

a Knockdown of IKKα or IKKβ, but not NF-κB, decreased ETS-1 expression. Cal27CP cells were transfected with non-target siRNA, IKKα, IKKβ, or p65 for 72 h and expression of IKKα, IKKβ, p65, ETS-1, and β-Actin were detected by Western blot analysis. b Both IKKα and IKKβ are involved in regulation of ETS-1 expression. Cal27CP cells were transfected with non-target siRNA, IKKα, IKKβ, or IKKα plus IKKβ for 72 h and expression of IKKα, IKKβ, ETS-1, and β-Actin were detected by Western blot analysis. c Knockdown of ETS-1 had no effects on p65 phosphorylation. Cal27CP cells were transfected with non-target siRNA or siRNA ETS-1 for 72 h and expression of IKKα, IKKβ, ETS-1, phospho-p65, p65, and β-actin were detected by Western blot.

Fig. 4. IKKβ associates with ETS-1 and regulates its degradation.

a The lysates from Cal27CP cells were immunoprecipitated with anti-IKKβ, anti-ETS-1, or IgG control, electrophoresed on an SDS gel, and detected with IKKβ and ETS-1 antibodies, respectively. Note: the red lines in panel A showed that blots were cut for detection of IKKβ (upper) and ETS-1 (bottom), respectively. b, c Cal27CP (b) and SCC25CP (c) cells treated with DMSO control or MG-132 for 2 h were incubated with media containing increasing concentrations of CmpdA for 24 h and lysed. The expression of ETS-1, p65, and β-actin was detected by Western blot analysis.

Fig. 5. Synergistic inhibition of IKKβ/NF-κB and ETS by combination of Dasatinib with IKKβ inhibitor, CmpdA.

Cal27CP (a) and SCC25CP (b) cells were treated with vehicle control, CmpdA, Dasatinib, or a combination for 24 h, lysed, and phosphorylation and total levels of SRC and p65 and expression of ETS-1, cleaved-caspase-3, and β-actin were detected by Western blot analysis.

Fig. 6. Synergistic induction of apoptosis after combination of Dasatinib with IKKβ inhibitor, CmpdA.

a–c Cal27CP (a) and SCC25CP (c) were treated with vehicle control, CmpdA, Dasatinib or a combination for 48 h. Cell apoptosis was measured by Annexin V. b–d Experiments in a and c were performed in triplicate, and early and late stage apoptosis in Cal27CP (b) and SCC25CP (d) were counted and statistical analysis was performed. P-values <0.05 were considered to be statistically significant.

Fig. 7. Dasatinib and CmpdA synergistically inhibit cell proliferation.

a, b Dasatinib and IKKβ inhibitor, CmpdA, synergistically inhibit cell proliferation. Cal27CP (a) or SCC25CP (b) cells were treated with DMSO, Dasatinib, CmpdA, or a combination for 72 h 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) were determined using CalcuSyn software. c, d Synergistic inhibition of colony formation by Dasatinib and CmpdA combination. Cal27CP (c) or SCC25CP (d) cells were treated with DMSO, Gefitinib, CmpdA, or a combination for 24 h and colony formation was observed 10 days after treatment. Each experiment was performed in triplicate.

Fig. 8. Model that illustrates pathways in response to Dasatinib and CmpdA treatment in cisplatin resistant-HNSCC cells.

Dasatinib inhibited SRC activity and lowered ETS-1 expression, but induced the IKK/NF-κB pathway, while the IKKβ inhibitor CmpdA blocked Dasatinib induction of IKK/NF-κB. A combination of Dasatinib and CmpdA led to significant inhibition of ETS-1 and IKK/NF-κB as well as cell proliferation and survival.