Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells

Abstract

Triple-negative breast cancer (TNBC) remains as an intractable malignancy with limited therapeutic targets. High expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis of TNBC; however, EGFR targeting has failed with unfavorable clinical outcomes. Here, we performed a combinatorial screening of fifty-five protein kinase inhibitors with the EGFR inhibitor gefitinib in the TNBC cell line MDA-MB-231 and identified the IκB kinase (IKK) inhibitor IKK16 as a sensitizer of gefitinib. Cell viability and clonogenic survival assays were performed to evaluate the antiproliferative effects of the gefitinib and IKK16 (Gefitinib + IKK16) combination in TNBC cell lines. Western blot analyses were also performed to reveal the potential mode of action of this combination. In addition, next-generation sequencing (NGS) analysis was performed in Gefitinib+IKK16-treated cells. The Gefitinib+IKK16 treatment synergistically reduced cell viability and colony formation of TNBC cell lines such as HS578T, MDA-MB-231, and MDA-MB-468. This combination downregulated p-STAT3, p-AKT, p-mTOR, p-GSK3β, and p-RPS6. In addition, p-NF-κB and the total NF-κB were also regulated by this combination. Furthermore, NGS analysis revealed that NF-κB/RELA targets including CCL2, CXCL8, EDN1, IL-1β, IL-6, and SERPINE1 were further reduced and several potential tumor suppressors, such as FABP3, FADS2, FDFT1, SEMA6A, and PCK2, were synergistically induced by the Gefitinib-+IKK16 treatment. Taken together, we identified the IKK/NF-κB pathway as a potential target in combination of EGFR inhibition for treating TNBC.

Keywords: IκB kinase (IKK) inhibition; anticancer; combination; epidermal growth factor receptor (EGFR) inhibition; gefitinib; kinase inhibitor; resistance; synergism; triple-negative breast cancer (TNBC).

Figure 1

Identification of IKK16 as a synergistic partner of gefitinib for anticancer activity in MDA-MB-231 cells. (A) The number of points with CI values greater than 1.3 and the mean CI values for these points are depicted. IKK16 was identified as a strong potentiator of gefitinib. The dashed lines indicate the thresholds for selection (x = 4.8 and y = 1.5, respectively. CI, classification index (see Section 2). (B) The MTT screening results for IKK16 in combination with gefitinib in MDA-MB-231 cells.

Figure 2

Treatment with IKK16 increased the sensitivity of TNBC cells to gefitinib. TNBC cells were treated with serially diluted concentrations of IKK16 in combination with gefitinib for 72 h. Cell viability was determined by MTT assay. (A–C) The combination effects of gefitinib with IKK16 in two MSL (HS578T and MDA-MB-231) and one BL1 (MDA-MB-468) type TNBC cells. Data are represented as the mean ± SD of results from at least three independent experiments performed in triplicates. ***, p < 0.001.

Figure 3

The Gefitinib+IKK16 combination reduced the survival of TNBC cells. HS578T, MDA-MB-231, and MDA-MB-468 cells were treated with 10 μM gefitinib, 2.5 μM IKK16, or the combination of 10 μM gefitinib and 2.5 μM IKK16 (Combo) for 24 h and cultivated for 10–14 days in normal growth media. The colonies were stained as described in the Materials and Methods. Representative images are shown from three independent experiments performed in triplicates. *, p < 0.05; ***, p < 0.001.

Figure 4

Inhibition of signaling pathways by the Gefitinib+IKK16 combination in TNBC cells. The cells were treated by drugs as indicated for 2 h (A) or 24 h (B), and the cell lysates were subjected to western blotting with antibodies against the proteins indicated. The β-tubulin was used as a loading control. Representative images were shown from three independent experiments.

Figure 5

Regulation of NF-κB p65 by the Gefitinib+IKK16 treatment. (A) Proteasomal degradation of NF-κB p65/RelA by the Gefitinib+IKK16 treatment in the absence or presence of MG132. The cells were treated with drugs for 24 h as indicated. MG132 (10 μM) was applied for 4 h before treatment of drugs. (B) Subcellular localization of NF-κB p65/RelA and RPS6. The cells were treated for 24 h and the cytoplasmic and nuclear fractions were subjected to western blot analysis. Either β-tubulin or Lamin B1 was used as a loading control. Representative images were shown from three independent experiments. (C) NF-κB reporter gene assay. HS578T cells transfected with the NF-κB reporter gene were treated with DMSO, gefitinib 5 μM), IKK16 (1.25 μM), or Gefitinib+IKK16 (5 μM and 1.25 μM, respectively) for 24 h and luciferase activities were determined. Data from three independent experiments are shown as the mean ± SEM. ***, p < 0.005.

Figure 6

Global transcriptional changes induced by the Gefitinib+IKK16 treatment. (A) Heat map of a total of 2403 genes identified that showed at least 2-fold change upon the gefitinib+IKK16 treatment in duplicated samples. (B) Heat map of 139 genes whose mRNA levels were reproducibly modulated by the gefitinib+IKK16 combination (≤mean ± 0.15). qRT-PCR results for (C) NF-κB/RELA target genes and (D) tumor suppressor genes in MDA-MB-231 cells treated with drugs for 24 h as indicated. Data from three independent experiments are shown as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.005.