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

Simple Summary Triple-negative breast cancer (TNBC) is an aggressive and intractable malignancy. Although a high-level expression of epidermal growth factor receptor (EGFR) is a distinct feature of TNBC, targeting EGFR has not been successful yet. Here, we described a combination of the EGFR inhibitor gefitinib and IκB kinase (IKK) inhibitor IKK16 (Gefitinib+IKK16) as a potential therapeutic approach to treat TNBC. The combination of these drugs resulted in reduced cell viability and survival in TNBC cells in vitro. Mechanistically, several components of PI3K/AKT/mTOR pathway were further downregulated by drug combination compared with single-agent treatments. Gene expression analysis revealed that several NF-κB/RELA targets were suppressed, and a couple of tumor suppressor genes were induced by the drug combination. Taken together, targeting IKK may potentiate EGFR inhibition in TNBC. 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.

The nuclear factor kappa light chain enhancer of activated B cells (NF-κB) is constitutively activated in most cancers, including TNBC, through various signaling pathways [28]. Activation of NF-κB is primarily regulated through its inhibitor, the inhibitor of NF-κB (IκB). Dissociation and subsequent degradation of phospho-IκB (p-IκB), which is mediated by IκB kinase (IKK) complex, leads to activation and nuclear translocation of the NF-κB transcription factor complex. In the nucleus, the NF-κB complex transactivates its target genes involved in immune regulation, anti-apoptosis, and cell proliferation [28][29][30].
The IKK complex has been demonstrated to play a crucial role in coupling inflammation and cancer [29]. The IKK complex consists of two catalytic subunits, IKKα (IKK1) and IKKβ (IKK2), and a regulatory subunit IKKγ (also known as NF-κB essential modulator, NEMO) [29]. These two catalytic subunits of IKK in humans have distinct roles in tumorigenesis; IKKβ has an NF-κB-dependent tumor-promoting functions, whereas IKKα has an NF-κB-independent role in tumor metastasis [29]. IKKα has been reported to be able to translocate into the nucleus to phosphorylate CREB-binding protein (CBP) leading to cancer development [31][32][33].
In this report, we demonstrated the potentiation of the EGFRi gefitinib by the IKK inhibitor IKK16 (also known as IKK Inhibitor VII) in human TNBC cell lines. Co-treatment with gefitinib and IKK16 (Gefitinib+IKK16) synergistically reduced viability of TNBC cells including HS578T, MDA-MB-231, and MDA-MB-468 cells. Long-term survival rates of these TNBC cells were also diminished by the Gefitinib+IKK16 treatment. The Gefitinib+IKK16 combination further inhibited the phosphorylation of the mammalian target of rapamycin (mTOR), glycogen synthase kinase-3 beta (GSK3β), and ribosomal protein S6 (RPS6) compared with the single treatments. Phosphorylation and nuclear translocation of NF-κB p65 was further inhibited, and inhibition of NF-κB transcriptional activity was enhanced by the Gefitinib+IKK16 combination. NGS analysis revealed the synergistic inhibition of NF-κB/RELA target genes and enhanced induction of potential tumor suppressors in response the Gefitinib+IKK16 combination.
All TNBC cells in this study were obtained from the American Type Culture Collection (Manassas, VA, USA). The cultured cells were monitor by trypan blue cell counting as described previously [58].

Clonogenic Survival Assay
TNBC cells, in 6-well plates, were treated with indicated drugs for 24 h, cultivated for 10-14 days in normal growth media, and the colonies were stained with crystal violet dissolved in a solubilizing buffer [1:1 mixture (v/v) of 0.1 M sodium phosphate buffer Cancers 2022, 14, 5215 4 of 21 (pH4.5) and ethanol] as previously described [20,22,27]. The number of colonies were determined after imaging the colonies using an image scanner.

Reporter Gene Assay
NF-κB-Luc vectors were previously described [61]. Cells were transiently transfected with NF-κB-Luc using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) on a 60-mm plate. One day after transfection, the cells were re-plated on 24 well plates (2 × 10 4 cells/well) and incubated overnight. Then the cells were treated with gefitinib, IKK16, or Gefitinib+IKK16 for 24 h. Luciferase assays were performed using a Dual-Luciferase ® Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Data are presented as the mean ± SEM of results from at least three independent experiments performed in triplicates.

Next-Generation Sequencing (NGS) Analysis
MDA-MB-231 cells were treated with vehicle control, gefitinib (3 µM), IKK16 (1.5 µM), or Gefitinib+IKK16 (3 µM and 1.5 µM, respectively) for 24 h in duplicates. NGS analysis was performed by Macrogen (Seoul, Korea). In brief, total RNA was isolated and treated with DNase. Ribosomal RNA (rRNA) was removed by ribo-zero rRNA removal kit. Isolated RNAs were randomly fragmented and converted to cDNA by reverse transcription. Prepared cDNAs with adapters were amplified by polymerase chain reaction (PCR) before sequencing. The raw transcriptome data was qualified by Phred quality score, trimmed, and further analyzed. For mapping cDNA fragments, genomic reference (GRCh38) was used.

Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) Analysis
Total RNA (1 µg) isolated from the treated cells was reverse transcribed to cDNA using PrimeScript first-strand cDNA synthesis kit (Takara Korea Biomedical Inc., Seoul, Republic of Korea) according to the manufacturer's instructions. Amplification of each cDNA was monitored using conf (PCR Biosystems Inc., Wayne, PA, USA) on a StepOnePlus instrument (Waltham, MA, USA). Specific primers used are listed in Table S1. Data are presented as the mean ± SEM of results from at least three independent experiments performed in triplicates.

Statistical Analysis
At least three independent experiments were performed in triplicate. Representative data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with a post-hoc Tukey's honest significance difference (HSD) test was used to compare differences between groups. Differences among groups were considered statistically significant when * p < 0.05, ** p < 0.01, and *** p < 0.005, respectively.

Identification of IKK16, an IKK Inhibitor, as a Potentiator of Gefitinib
We have performed a cell viability screening of protein kinase inhibitors (PKIs) in combination with the EGFR inhibitor gefitinib in a human TNBC cell line, MDA-MB-231. As reported previously [19,22,27,[62][63][64][65][66][67][68], various PKIs targeting the phosphoinositide-3kinase (PI3K)/AKT/mTORC1 pathway have been repeatedly identified as potentiators of gefitinib, implicating that this pathway may contribute to intrinsic resistance of TNBC to EGFRis (Figure 1 and Table 1). Among them, AT7867, an inhibitor of AKT/p70 S6 kinase (p70S6K)/protein kinase A (PKA), was previously reported as a promising PKI to potentiate anticancer activity of gefitinib in MDA-MB-231 cells [27]. In this study, we analyzed the previous screening results further, and found that our new analysis more clearly revealed the potential candidates of co-treatment with gefitinib in MDA-MB-231 cells ( Figure 1A). IKK16 alone was relatively potent to reduce the viability of MDA-MB-231 cells compared with gefitinib or TPCA-1 ( Figure 1A Combination of IKK16 with gefitinib resulted in a synergistic antiproliferative effect in multiple combination points ( Figure 1B).

Statistical Analysis
At least three independent experiments were performed in triplicate. Representa data are presented as the mean ± standard deviation (SD). One-way analysis of varia (ANOVA) with a post-hoc Tukey's honest significance difference (HSD) test was use compare differences between groups. Differences among groups were considered st tically significant when * p < 0.05, ** p < 0.01, and *** p < 0.005, respectively.

Identification of IKK16, an IKK Inhibitor, as a Potentiator of Gefitinib
We have performed a cell viability screening of protein kinase inhibitors (PKIs combination with the EGFR inhibitor gefitinib in a human TNBC cell line, MDA-MB-As reported previously [19,22,27,[62][63][64][65][66][67][68], various PKIs targeting the phosphoinositid kinase (PI3K)/AKT/mTORC1 pathway have been repeatedly identified as potentiator gefitinib, implicating that this pathway may contribute to intrinsic resistance of TNB EGFRis ( Figure 1 and Table 1). Among them, AT7867, an inhibitor of AKT/p70 S6 kin (p70S6K)/protein kinase A (PKA), was previously reported as a promising PKI to po tiate anticancer activity of gefitinib in MDA-MB-231 cells [27]. In this study, we analy the previous screening results further, and found that our new analysis more clearly vealed the potential candidates of co-treatment with gefitinib in MDA-MB-231 cells ( ure 1A). IKK16 alone was relatively potent to reduce the viability of MDA-MB-231 c compared with gefitinib or TPCA-1 ( Figure    Interestingly, the protein kinase C (PKC) inhibitors, such as Ro-31-8220, chelerythrine, and enzastaurin, were identified as potential candidates for gefitinib potentiation in MDA-MB-231 cells ( Figure 1 and Table 1). In addition, imatinib, which had previously been reported as a combinational treatment partner of the dual EGFR/human EGFR receptor 2 (HER2) inhibitor lapatinib [93], was also identified from the screening. Imatinib is an US FDA-approved PKI for the treatment of rare gastrointestinal cancer and acute lymphocytic leukemia [13]. Another interesting feature of the screening results is that PKIs targeting the aurora kinases (AURKs) were found to be potentiators for gefitinib. AZD1152 (barasertib), danusertib (PHA-739358), and ZM-447439 are targeting AURKs (Table 1). PKIs, targeting kinases involved in DNA damage repair, have been in the list of candidates, such as the DNA-dependent protein kinase (DNA-PK) and checkpoint kinase 1 (CHEK1). Protein kinases, such as adenosine monophosphate-activated protein kinase (AMPK), Janus kinase 2 (JAK2), and P38α were also identified as potential targets to synergize gefitinib efficacy in MDA-MB-231 cells. None of these protein kinases have been reported previously as potential targets for EGFRi potentiation [14].
Among these PKIs, we selected IKK16 for further investigation. IKK16 is a selective IKK inhibitor for IKKβ, IKK complex, and IKKα with IC 50 values of 40, 70, and 200 nM, respectively [78]. TPCA-1 (GW683965) is also a selective inhibitor of IKKβ with an IC 50 value of 17.9 nM [88], and the potency of TPCA-1 in combination with gefitinib was relatively low ( Figure 1A). Notably, TPCA-1 has been also reported as an inhibitor of Janus kinase 1 (JAK1) with an IC 50 value of 43.78 nM [89]. To the best of our knowledge, this is the first report on synergistic efficacy of the combination of gefitinib and an IKK inhibitor in TNBC cells. The synergistic effects of the Gefitinib+IKK16 combination were further accessed in two mesenchymal stem-like (MSL) cell lines, HS578T and MDA-MB-231, and a basal-like 1 (BL1) cell line, MDA-MB-468. The combination of gefitinib and IKK16 in a 4-to-1 ratio dramatically reduced the viability of all TNBC cell lines tested (Figure 2). A complete loss of viability was observed with 2.25 µM gefitinib combined with 9 µM IKK16 in HS578T and MDA-MB-231 cells, and with 1.5 µM gefitinib combined with 6 µM IKK16 in MDA-MB-468 cells. Similar to previous findings [19], the BL1 TNBC cell line MDA-MB-468 was more sensitive to gefitinib, IKK16, and their combination. Collectively, the Getifinib+IKK16 combination synergistically reduced the viability of TNBC cells in vitro.
Cancers 2022, 14, x 7 of 22 kinase 1 (JAK1) with an IC50 value of 43.78 nM [89]. To the best of our knowledge, this is the first report on synergistic efficacy of the combination of gefitinib and an IKK inhibitor in TNBC cells.
The synergistic effects of the Gefitinib+IKK16 combination were further accessed in two mesenchymal stem-like (MSL) cell lines, HS578T and MDA-MB-231, and a basal-like 1 (BL1) cell line, MDA-MB-468. The combination of gefitinib and IKK16 in a 4-to-1 ratio dramatically reduced the viability of all TNBC cell lines tested (Figure 2). A complete loss of viability was observed with 2.25 μM gefitinib combined with 9 μM IKK16 in HS578T and MDA-MB-231 cells, and with 1.5 μM gefitinib combined with 6 μM IKK16 in MDA-MB-468 cells. Similar to previous findings [19], the BL1 TNBC cell line MDA-MB-468 was more sensitive to gefitinib, IKK16, and their combination. Collectively, the Getifinib+IKK16 combination synergistically reduced the viability of TNBC cells in vitro.

Inhibition of Long-Term Survival of TNBC Cells by the Gefitinib+IKK16 Treatment
Since MTT assay results are not enough to determine the anticancer drug activity on the inhibition of proliferation and survival of residual cancer cells [94][95][96][97], we further analyzed the Gefitinib+IKK16 activity using a clonogenic assay. TNBC cells were treated with drug combinations for 24 h and further cultivated in normal growth medium without drugs for 10-14 days. As reported previously [19,20,22,27], gefitinib alone did not reduce the number of colonies from three TNBC cell lines (Error! Reference source not found.). Interestingly, IKK16 itself significantly inhibited the colony formation in all TNBC cell lines tested. In addition, the survival rates of TNBC cell lines were further reduced by the Gefitinib+IKK16 treatment. Marked reduction in the colony numbers was found in HS578T and MDA-MB-468 cells.

Inhibition of Long-Term Survival of TNBC Cells by the Gefitinib+IKK16 Treatment
Since MTT assay results are not enough to determine the anticancer drug activity on the inhibition of proliferation and survival of residual cancer cells [94][95][96][97], we further analyzed the Gefitinib+IKK16 activity using a clonogenic assay. TNBC cells were treated with drug combinations for 24 h and further cultivated in normal growth medium without drugs for 10-14 days. As reported previously [19,20,22,27], gefitinib alone did not reduce the number of colonies from three TNBC cell lines ( Figure 3). Interestingly, IKK16 itself significantly inhibited the colony formation in all TNBC cell lines tested. In addition, the survival rates of TNBC cell lines were further reduced by the Gefitinib+IKK16 treatment. Marked reduction in the colony numbers was found in HS578T and MDA-MB-468 cells.  Figure 4B). To date, no previous study has reported the regulation of p-STAT3 (Y705) by IKK. The phosphorylation of tyrosine 705 residue is mediated by JAK and SRC leading to activation of STAT3 [98]. It is worthy to note that stabilization of IKKα by direct physical interaction of STAT3 has been reported to prevent its proteasomal degradation, leading to activation of non-canonical NF-κB pathway during tumorigenesis of breast epithelial cells [99]. Cancers 2022, 14, x 8 of 22

Downregulation of p-STAT3, p-AKT, p-mTOR, p-GSK3β, p-RPS6 in TNBC Cells by the Gefitinib+IKK16 Treatment
Changes in the levels of signaling pathway components of interest were accessed by western blot analysis in two TNBC cell lines. HS578T and MDA-MB-231 cells were treated with drug combinations for 2 h or 24 h in normal growth media. No significant changes in the levels of p-STAT3 (Y705) in response to gefitinib treatment alone was observed (Figure 4A). Interestingly, IKK16 weakly reduced the level of p-STAT3 (Y705) in both cell lines 2 h post treatment. The Gefitinib+IKK16 combination further reduced the levels of p-STAT3 (Y705). The inhibition was pertained to 24 h ( Figure 4B). To date, no previous study has reported the regulation of p-STAT3 (Y705) by IKK. The phosphorylation of tyrosine 705 residue is mediated by JAK and SRC leading to activation of STAT3 [98]. It is worthy to note that stabilization of IKKα by direct physical interaction of STAT3 has been reported to prevent its proteasomal degradation, leading to activation of non-canonical NF-κB pathway during tumorigenesis of breast epithelial cells [99]. The phosphorylation of AKT (S473) was reduced by gefitinib alone and further reduced by the Gefitinib+IKK16 combination in HS578T cells treated for 2 h. However, the gefitinib-mediated inhibition of p-AKT was abolished in HS578T cells treated for 24 h. This recurrence of p-AKT was partially suppressed by Gefitinib+IKK16 treatment in HS578T cells ( Figure 4B). As previously reported, the levels of p-AKT (S473) were barely detectable in MDA-MB-231 cells cultured in normal growth media containing 10% FBS [16,17,19,20,22].
The levels of p-GSK3β (S9), the downstream target of AKT, were reduced by gefitinib alone. This inhibitory effect was more apparent in TNBC cells treated for 2 h. The Gefi-tinib+IKK16 combination further reduced p-GSK3β (S9) levels for up to 24 h. Interestingly, IKK16 reduced the levels of p-GSK3β (S9) in TNBC cells treated for 2 h, but not in those treated for 24 h ( Figure 4B).
Treatment with gefitinib or IKK16 alone had little or no effect on the levels of p-mTOR (S2448), another AKT downstream effector. However, the Gefitinib+IKK16 treatment reduced p-mTOR (2448) levels in both cell lines. Most interestingly, the Gefitinib+IKK16 combination synergistically reduced the p-RPS6 (S235/236) levels, which were inhibited by individual treatments with both gefitinib or IKK16 in HS578T cells. The levels of total RPS6 were also marginally reduced by the Gefitinib+IKK16 treatment. It has been reported Cancers 2022, 14, 5215 9 of 21 that siRNA-based knockdown of RPS6 was sufficient to reduce the viability of TNBC cells [20], suggesting RPS6 as a potential target for treating cancer [100]. Taken together, the Gefitinib+IKK16 combination suppressed the PI3K/AKT/mTOR pathway in TNBC cells. The phosphorylation of AKT (S473) was reduced by gefitinib alone and further reduced by the Gefitinib+IKK16 combination in HS578T cells treated for 2 h. However, the gefitinib-mediated inhibition of p-AKT was abolished in HS578T cells treated for 24 h. This recurrence of p-AKT was partially suppressed by Gefitinib+IKK16 treatment in HS578T cells ( Figure 4B). As previously reported, the levels of p-AKT (S473) were barely detectable in MDA-MB-231 cells cultured in normal growth media containing 10% FBS [16,17,19,20,22].
The levels of p-GSK3β (S9), the downstream target of AKT, were reduced by gefitinib alone. This inhibitory effect was more apparent in TNBC cells treated for 2 h. The Ge-fitinib+IKK16 combination further reduced p-GSK3β (S9) levels for up to 24 h. Interestingly, IKK16 reduced the levels of p-GSK3β (S9) in TNBC cells treated for 2 h, but not in those treated for 24 h ( Figure 4B).
Treatment with gefitinib or IKK16 alone had little or no effect on the levels of p-mTOR (S2448), another AKT downstream effector. However, the Gefitinib+IKK16 treatment reduced p-mTOR (2448) levels in both cell lines. Most interestingly, the Ge-fitinib+IKK16 combination synergistically reduced the p-RPS6 (S235/236) levels, which were inhibited by individual treatments with both gefitinib or IKK16 in HS578T cells. The levels of total RPS6 were also marginally reduced by the Gefitinib+IKK16 treatment. It has

Regulation of NF-κB by the Gefitinib+IKK16 Treatment in TNBC Cells
Since IKK regulates the stability of IκB and localization of NF-κB, we further assessed the changes in the levels of these proteins by western blotting. Unlike lapatinib [101], gefitinib treatment did not induce increases in p-NF-κB p65 levels ( Figure 5A). As expected, the levels of IκB were increased by IKK16 treatment in HS578T and MDA-MB-231 cells ( Figure 5A). Inhibition of IκB phosphorylation was evidenced in cells treated with the proteasome inhibitor MG132 [102]. Blocking IKK activities resulted in the reduction of p-NF-κB levels.
Nuclear accumulation of NF-κB p65/RelA was further evaluated in HS578T and MDA-MB-231 cells treated with IKK16 ( Figure 5B). Previously, it has been reported that dephosphorylation of p-NF-κB p65 (S536) occurs in the nucleus [103]. A slight increase in NF-κB p65 was observed in cells treated with IKK16. The Gefitinib+IKK16 treatment reduced this increase in nuclear NF-κB p65 levels. However, the role of p-NF-κB p65 (S536) in tumorigenesis and cancer progression remains to be determined [104].
Since IKK regulates the stability of IκB and localization of NF-κB, we further assessed the changes in the levels of these proteins by western blotting. Unlike lapatinib [101], gefitinib treatment did not induce increases in p-NF-κB p65 levels ( Figure 5A). As expected, the levels of IκB were increased by IKK16 treatment in HS578T and MDA-MB-231 cells ( Figure 5A). Inhibition of IκB phosphorylation was evidenced in cells treated with the proteasome inhibitor MG132 [102]. Blocking IKK activities resulted in the reduction of p-NF-κB levels.  Figure 5B). Previously, it has been reported that dephosphorylation of p-NF-κB p65 (S536) occurs in the nucleus [103]. A slight increase in Unexpectedly, the extent of nuclear localization of p-RPS6 (S235/236) differed between HS578T and MDA-MB-231 cells ( Figure 5B). The p-RPS6 was evenly localized in both cytoplasm and nucleus in HS578T cells, whereas an exclusively cytoplasmic localization of p-RPS6 was observed in MDA-MB-231 cells. The significance of this discrepancy remains elusive.
Since the levels of nuclear NF-κB p65 were reduced by the Gefitinib+IKK16 treatment, we further performed NF-κB reporter gene assays in HS578T cells ( Figure 5C). For reporter gene assay, the concentration of EC 50 for the Gefitinib+IKK16 treatment was selected. As expected, treatment with IKK16 alone markedly reduced the luciferase activity in HS578T cells. Little or no effect of gefitinib on the luciferase activity was observed. However, most profound reduction of the luciferase activity was achieved by the Gefitinib+IKK16 combination. Taken together, these data support that the transcriptional activity of NF-κB was synergistically reduced by the Gefitinib+IKK16 combination in HS578T cells.

Transcriptomic Regulation by the Gefitinib+IKK16 Treatment
The global effect on the transcriptional regulation induced by the Gefitinib+IKK16 (10 µM and 2.5 µM, respectively) combination was analyzed by NGS in MDA-MB-231 cells. A total of 2403 genes were identified that exhibited at least 2-fold change (fc2) in the duplicated samples ( Figure 6A). We further narrowed down the list to 139 genes with mRNA levels reproducibly modulated by drug treatment (≤mean ± 0.15) in duplicates ( Figure 6B). Twenty-three genes were identified as targets for NFKB1 or RELA by the ConsensusPathDB (http://cpdb.molgen.mpg.de/, accessed on 10 May 2022) [105] or reference analysis. To further confirm their enhanced regulation by the Gefitinib+IKK16 combination, we performed qRT-PCR analysis of selected gene transcripts in RNA samples from MDA-MB-231 cells treated with gefitinib (10 μM), IKK16 (2.5 μM), or the Gefitinib+IKK16 combination (10 μM and 2.5 μM, respectively) for 24 h (Table 2).  To further confirm their enhanced regulation by the Gefitinib+IKK16 combination, we performed qRT-PCR analysis of selected gene transcripts in RNA samples from MDA-MB-231 cells treated with gefitinib (10 µM), IKK16 (2.5 µM), or the Gefitinib+IKK16 combination (10 µM and 2.5 µM, respectively) for 24 h (Table 2).  [118] In the qRT-PCR analysis, a subset of the NF-κB/RELA target genes, such as CCL2, CXCL8, EDN1, IL-1β, IL-6, and SERPINE1, was identified as targets of the Gefitinib+IKK16 treatment ( Figure 6C). Generally, gefitinib alone reduced the mRNA levels of these genes, but IKK16 had more profound inhibitory effects. The Gefitinib+IKK16 combination further enhanced the gefitinib-mediated suppression of these genes. As shown in Table 2, the genes identified as targets of the combinatory treatment have important roles in tumorigenesis, and inhibition of their function induces anti-tumor effects.
Unexpectedly, the transcript levels of two RELA target genes were upregulated by the Gefitinib+IKK16 combination. One of these, PCK2, has been reported as a tumor suppressor in renal cell carcinoma [112]. The other one, TRIB3, is a master oncogenic factor. The mechanisms of transcriptional regulation of these gene have not been understood yet. In addition, it would be noteworthy that the mRNA expression of TRIB3 was markedly enhanced by the Gefitinib+IKK16 combination ( Figure 6C). Although the Gefitinib+IKK16 combination showed synergistic anticancer effects, the induction of an oncogenic factor by this combination still warrants the need for further investigation of the mechanisms and/or additional drug combinations to overcome potential recurrence of drug resistance.
Interestingly, the mRNA expression levels of several potential tumor suppressor genes were markedly increased by the Gefitinib+IKK16 combination ( Figure 6D). However, the underlying molecular mechanisms of this synergistic induction remain to be investigated. Taken together, the addition of IKK16 overcomes the resistance of TNBC cells to gefitinib, potentially through the transcriptional inhibition of the NF-κB/RELA target genes and induction of a set of tumor suppressor genes in TNBC cells.
Previously, an inverse correlation has been reported in the levels of EGFR and ER in ER(-) and ER positive BC cells [122][123][124][125]. In addition, as revealed by a comparative study, EGF is a major autonomous growth-promoting factor for TNBC cells [41]. Furthermore, the level of active NF-κB in TNBC cells are elevated by the EGF-EGFR axis and inhibited by the anti-EGFR antibody and the PKC inhibitor Go6976 [41]. The activated NF-κB transactivates the cell-cycle regulator, cyclin D1, leading to increase of p-retinoblastoma (p-RB) in ER(-) cells in a PI3K/PKC/IKK-dependent manner [41]. Constitutive activation of NF-κB has also been reported in TNBCs [126,127]. Blocking activated NF-κB by small-molecule inhibitor or by expression of IκBα super-repressor leads to apoptotic cell death or reduced proliferation of TNBC cells [41,126]. More interestingly, an adaptive survival program is induced by EGFR inhibition, leading to rapid formation of the EGFR-tumor necrosis factor receptorassociated factor 2 (TRAF2)-receptor-interacting protein 1 (RIP1)-IKK complex to activate NF-κB in non-small cell lung cancer xenograft model [128].
NGS analysis and subsequent qRT-PCR analysis suggested that blocking IKK potentiates EGFR inhibition at least partially though the suppression of NF-κB/RELA-dependent transcription of tumorigenic genes and induction of tumor suppressors. Since IKK16 also inhibits IKKα which has an NF-κB-independent role, regulation of tumor suppressor genes may be mediated by EGFR-IKKα in TNBC cells. Interestingly, IKKα-dependent activation of NOTCH1 signaling has been reported to induce p-AKT, oxidative metabolism, and transcriptional activation of survival genes in PTEN wild-type TNBC cells [150].
In summary, the present study provides evidence that supports the combined targeting of EGFR and IKK as a potential therapeutic strategy for TNBC treatment.

Conclusions
In the present study, we identified the IKK/NF-κB pathway as a potential target for enhancing the efficacy of EGFR inhibition in TNBC cells. Combined inhibition of this pathway with EGFR inhibition resulted in the reduction in cell viability and longterm survival of TNBC cells. NGS analysis revealed that a subset of the NF-κB/RELA target genes was synergistically suppressed, and expression levels of a series of tumor suppressor genes were elevated by the co-targeting EGFR and IKK with specific small molecule inhibitors. These results warrant further studies on the therapeutic potential of targeting the IKK/NF-κB pathway in combination with current therapeutics for treating TNBC in the future.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers14215215/s1, Table S1: Primers used for qRT-PCR. File S1: Original WB Images. Data Availability Statement: Data in this study will be available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.