Introduction
IκB kinase ε (IKKε, also named IKKi/IKBKE) is a member of the IKK family, which contains five distinct but closely related members: IKKα, IKKβ, IKKγ, TBK-1 and IKKε [
1,
2]. IKK is an important mediator of the activation of NF-κB, which is a heterodimeric transcription factor playing essential roles in inflammation and cancer pathogenesis. The NF-κB family is composed of Rel A, Rel B, c-Rel, p50/p105 and p52/p100. Inhibitors of kappa B (IκBs) bind to the homodimers or heterodimers of NF-κB proteins and cause their cytoplasmic retention in an inactivated form. Upon stimulation, IκBs are phosphorylated by IKK complexes - which leads to the ubiquitination and proteasomal degradation of IκBs. NF-κB is then released and translocated into the nucleus to regulate the expression of target genes involved in immune and inflammatory responses [
3,
4]. Discovered in 2000, IKKε shows a 33% and 31% sequence identity with IKKα and IKKβ, respectively, in the N-terminal kinase domain, but has distinct function in the activation of NF-κB pathway [
2,
5]. Overexpression of IKKε is strongly correlated with the nuclear localization of c-Rel in breast cancer specimens, indicating that a substantial fraction of NF-κB activation is induced by aberrant IKKε in breast cancer cells [
6]. The relationship between IKKε and NF-κB, however, is not fully understood [
4,
7].
IKKε is primarily involved in signaling of inflammatory and immune processes [
8,
9]. Peant and colleagues reported that overexpression of IKKε in hormone-sensitive LNCaP and 22Rv1 prostate tumor cells induced secretion of numerous inflammatory cytokines, such as IL-8 and IL-6. However, the IKKε-dependent IL-8 and IL-6 overexpressions are not mediated by the activation of NF-κB pathway. Instead, the authors speculated that high IKKε expression leads to nuclear translocation of itself to activate these inflammatory cytokine genes [
10]. Recently, the role of IKKε in cancer has been studied by several groups. Sonenshein and colleagues observed for the first time a higher level of IKKε in breast cancer cell lines and specimens, whereas little IKKε expression was detected in normal breast epithelial cells [
11]. Furthermore, Boehm and colleagues indentified IKKε as a new potential oncogene in breast cancer cell lines and patient-derived tumors using three complementary genetic approaches. Overexpression of IKKε was observed in over 30% of breast cancer cell lines and carcinomas [
4,
6,
7]. On the other hand, inhibition of IKKε in breast cancer cells with overexpressed IKKε induced cell death [
6]. All these up-to-date data strongly support the role of IKKε in tumorigenesis, and subsequently blocking the IKKε expression would be a rational strategy to treat breast cancer.
Among various strategies to inhibit the oncogene expression, RNA interference (RNAi) offers considerable promise for cancer therapy due to its ability to potently knockdown a specific gene. siRNA of 21 to 23 nucleotides in length silences a target gene by binding to its complementary mRNA and triggering its degradation [
12,
13]. In the present study, we intend to evaluate the effect of silencing IKKε on colonigenicity, invasive properties, proliferation, and apoptosis in breast cancer cells using synthetic siRNA.
Materials and methods
Reagents
Lipofectamine-2000 and TRIzol reagent were purchased from Invitrogen Corp. (Carlsbad, CA, USA), Cell culture products were obtained from Atlanta Biologicals, Inc. (Lawrenceville, GA, USA) and Mediatech, Inc. (Manassas, VA, USA). BSA was purchased from Sigma-Aldrich Corporation (St Louis, MO, USA). SYBR Green-1 dye universal master mix and Multiscript RT were purchased from Applied Biosystems, Inc. (Foster City, CA, USA). The 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate Membrane Insert was purchased from Corning Incorporated (Lowell, MA, USA). BD Matrigel™ and BD Pharmingen™ Annexin V-FITC Apoptosis Detection Kit I was obtained from BD Biosciences (San Jose, CA, USA). The CellTiter-Glo® Luminescent Cell Viability Assay Kit was purchased from Promega Corp. (Madison, WI, USA). The NF-κB-Met-Luc2 reporter vector was obtained from Clontech Laboratories, Inc. (Mountain View, CA, USA). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Research Products International Corp. (Mt. Prospect, IL, USA). Cisplatin was obtained from Enzo Life Sciences, Inc. (Plymouth Meeting, PA, USA). Doxorubicin hydrochloride was purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA).
Cell lines and culture conditions
All cell lines (human breast cancer cell lines, SK-BR-3 and MCF-7) were purchased from the American Type Culture Collection, and were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 unit/ml), and streptomycin (100 μg/ml). Both cell lines were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was changed every other day and the cells were passaged when they reached 80 to 90% confluency.
siRNA design and synthesis
siRNAs targeting IKKε [GenBank: NM_014002] were designed using BLOCK-iT™ RNAi Designer (Invitrogen), siRNA Target Finder (Ambion, Austin, TX, USA), siRNA Target Finder (GeneScript, Piscataway, NJ, USA) and siRNA target Designer (Promega). Eight siRNAs targeting different regions of IKKε mRNA were designed (Table
1) and were purchased from Ambion and Invitrogen. These synthetic siRNAs are of 19 nucleotides with two thymidine deoxynucleotide (T) 3' overhangs. All designed siRNA sequences were blasted against the human genome database to eliminate cross-silence phenomenon with nontarget genes. Scrambled siRNA (Ambion) that does not target any gene was used as the negative control siRNA.
Table 1
Sense strand sequence of IKKε siRNA [GenBank: NM_014002]
siR-1 | 482 | 5'-GGUCUUCAACACUACCAGCtt-3' |
siR-2 | 2538 | 5'-GGCAUCCUGAAGCAUUAGAtt-3' |
siR-3 | 551 | 5'-GCUGAACCACCAGAACAUCtt-3' |
siR-4 | 533 | 5'-GUUUGAGGUCCUGCGGAAGtt-3' |
siR-5 | 820 | 5'-GCAUCUACAAGCUGACAGAtt-3' |
siR-6 | 1960 | 5'-GGGAUCAGGUACAUGAGGAtt-3' |
siR-7 | 1968 | 5'-GUACAUGAGGACAGAAGCAtt-3' |
siR-8 | 1978 | 5'-ACAGAAGCAUCCAGCAGAUtt-3' |
Transfection of siRNA
Cells were transfected with siRNA and Lipofectamine-2000 according to the manufacturer's instructions. Briefly, cells were seeded in a 24-well-plate at a density of 0.5 × 105 cells/well with antibiotics-free medium 12 hours before the transfection. One and a half microliters of the siRNA (20 μM) were mixed with 1 μl Lipofectamine-2000 in 50 μl serum-free RPMI-1640 medium and were incubated at room temperature for 25 minutes to form a complex. After washing cells with PBS, the 50 μl transfection mixtures were added to each well with 450 μl RPMI-1640 medium containing 10% FBS at a final concentration of 50 nM siRNA. Twenty-four hours after the transfection, the medium was replaced with fresh 500 μl RPMI-1640 medium containing 10% FBS. Forty-eight hours after the transfection, cells were collected for RNA and protein isolation.
Real-time RT-PCR
Total RNA was isolated from cells using TRIzol reagent according to the manufacturer's protocol. Total RNA (200 ng) was converted to cDNA using random hexamer primer and MultiScribe Reverse Transcriptase Reagent. One hundred nanograms of cDNA were amplified by real-time PCR using SYBR Green-1 dye universal Master mix on an ABI Prism 5700 Sequence Detection System (Applied Biosystems). To confirm the PCR specificity, PCR products were subjected to a melting-curve analysis. The comparative threshold method was used to calculate the relative amount of mRNA of treated sample in comparison with control samples [
14,
15]. The primers used for the study included: IKKε, 5'-ACTCTGGAAGTGGCAA GGACAT-3' (forward) and 5'-TACCTGATCCCGGCTCTTCACCA-3' (reverse); IKKα, 5'-TCT GGAACAGCGTGCCATTGATCT-3' (forward) and 5'-ATTACTGAGGGCCACTTCCACCTT-3' (reverse); IKKβ, 5'-ACTGGAGCAGCAGAAGTACACAGT-3' (forward) and 5'-ATCAG CATCAGTTGCAGCCACTTC-3' (reverse); TBK1, 5'-AGGATTGCCTGATCCAGCCAAGAT-3' (forward) and 5'-CCACTGGACGAAGGAAGCTTATGC-3' (reverse); and Bcl-2, 5'-AGGCAT GTTGACTTCACTTGTGGC-3' (forward) and 5'-GCATGCGGCCTCTGTTTGATTTCT-3' (reverse). We used 18s ribosomal RNA as an internal control, and the primers were 5'-GTCTGTGATGCCCTTAGATG-3' (forward primer), and 5'-AGCTTATGACCCGCACTT AC-3' (reverse primer).
Western blotting
The cultured cells were washed twice with ice-cold PBS and lysed on ice in RIPA lysis buffer containing freshly added protease and phosphatase inhibitor cocktails. After 5 minutes of incubation, the cell lysate was collected by centrifugation at 4°C for 10 minutes at 12,000 rpm. The amount of total protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of total protein (20 μg) was loaded and separated by SDS-PAGE. The protein was transferred to a nitrocellulose membrane, blocked and probed with appropriate antibodies. The protein was then visualized using horseradish peroxidase-conjugated secondary antibodies and the FluorChem FC2 imaging system (Alpha Innotech, San Leandro, CA, USA). Anti-IKKε/IKKi antibody (Sigma-Aldrich), anti-β-Actin antibody (Rockland, Gilbertsville, PA, USA), anti-Bcl2 antibody (Abcam, Cambridge, MA, USA), anti-cyclin D1 (Abcam) and horseradish peroxidase-conjugated secondary antibody (Invitrogen) were used in the western blotting assay.
Forty-eight hours after the transfection, 5 × 103 MCF-7 cells/well or 7.5 × 103 SK-BR-3 cells/well were seeded in six-well plates. The medium was changed every 2 days. Cells cultured for 9 days were washed twice with ice-cold medium, fixed by ice-cold methanol, and stained with 0.2% crystal violet. Images of the colonies were obtained using a digital camera.
Soft agar assay
Colony formation ability was examined by anchorage-independent soft agar assay on MCF-7 cells. Briefly, 1.5 ml FBS supplemented medium containing 0.5% agarose were added in 35-mm cell culture dishes and allowed to solidify (base agar). Next, 1 × 104 siRNAs transfected MCF-7 cells were mixed with 1.5 ml FBS-supplemented medium containing 0.35% agarose and added to the top of base agar. The cells were then cultured for 14 days at 37°C under 5% carbon dioxide. The dishes were stained with 0.005% crystal violet, and the colonies were examined with microscope and digital camera.
Wound healing assay
SK-BR-3 cells seeded in 12-well plates (2 × 10
5 cells/well) were transfected with 50 nM siRNA as described above. Once the cells reached 90% confluency, a wound area was carefully created by scraping the cell monolayer with a sterile 10 μl pipette tip. The cells were then washed once with Dulbecco's PBS to remove detached cells. Subsequently, the cells were incubated at 37°C in 5% carbon dioxide. The width of the wound area was monitored with an inverted microscope at various time points. The normalized wound area (wound area
48 hours/wound area
0 hours) was calculated using the software TScratch [
16].
Migration assay and invasion assay
We evaluated the effect of IKKε siRNA on invasiveness properties of breast cancer cells using transwell migration and invasion assays. Forty-eight hours after the transfection, SK-BR-3 cells or MCF-7 cells were trypsinized and resuspended in FBS-free RPMI-1640 medium. For the migration assay, a total of 1 × 10
5 cells were plated in the top chamber of the transwell with a noncoated polycarbonate membrane (6.5 mm diameter insert, 8.0 μm pore size; Corning Incorporated). For the invasion assay, 1 × 10
5 cells were plated in the top chamber of the transwell with a matrigel-coated polycarbonate membrane. RPMI-1640 medium with 10% FBS was added to the lower chamber as a chemoattractant. After incubation for 48 hours (migration assay) or 60 hours (invasion assay), cells on the lower surface of the membrane were fixed with 10% formalin and stained with 0.2% crystal violet. Cells that did not migrate through the pores were mechanically removed by a cotton swab [
17]. The images of migrated cells were acquired by an inverted microscope with a magnification of 200×. The number of migrated or invaded cells was counted from five or six randomly selected fields in a blind way.
Cell proliferation assay
The effect of siRNA on cell proliferation was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's protocol. Briefly, SK-BR-3 cells (5,000 cells/well) or MCF-7 cells (2,500 cells/well) seeded in 96-well plates were transfected with 50 nM siRNA as described above. Seventy-two hours and 120 hours after the transfection, 100 μl CellTiter-Glo® reagent was added to each well that contained 100 μl cell culture medium. Cells were then lysed by shaking in an orbital shaker for 2 minutes, followed by incubation at room temperature for 10 minutes to stabilize the luminescent signal. The luminescent intensity was measured using a Beckman DTX 880 multimode Detector (Beckman coulter, Inc., Brea, CA, USA).
NF-κB transcriptional activity assay
The transcriptional activity of NF-κB was examined using a Ready-To-Glow™ secreted luciferase reporter system, NF-κB-Met-Luc2, which contains the NF-κB promoter element upstream of the luciferase gene. The expression of luciferase was used to monitor the activity of NF-κB. Fifty thousand SK-BR-3 cells or MCF-7 cells were seeded in 24-well plates and transfected with siRNAs. Twenty-four hours after siRNA transfection, the cells were co-transfected with NF-κB-Met-Luc2 reporter vector and β-galactosidase reporter vector (used as an internal control). The culture medium was collected 24 hours post-transfection to measure the luciferase activity. The cells were lysed with reporter lysis buffer and the β-galactosidase activities of whole cell lysate were measured. The relative luciferase activity was calculated by normalizing results with the β-galactosidase expression.
Cell cycle assay and apoptosis assay
Forty-eight hours after the siRNA transfection, the cells were collected and fixed with ice-cold 70% ethanol. Before staining, the cells were washed with Dulbecco's PBS and incubated with propidium iodide/RNase staining buffer for 30 minutes at room temperature. Cell cycle analysis was carried out with a FACSCalibur Flow cytometer (BD Biosciences). To analyze apoptosis, cells were collected 72 hours post-transfection, and then stained with Annexin V-FITC and propidium iodide using the Annexin V-FITC Apoptosis Detection Kit I. The percentage of apoptotic cells was quantified by a FACSCalibur Flow cytometer. Paclitaxel (100 nM, 24-hour incubation) was used in the apoptosis assay as an apoptosis inducer to validate the measurements.
Combinational treatment of cells with siRNA and chemotherapy agents
Cells (10,000 cells/well) were seeded in a 96-well plate and transfected with siRNA as described above. Twenty-four hours after the transfection, the cells were incubated with medium supplemented with serial concentrations of cisplatin and doxorubicin for another 24 hours. Untransfected cells treated with different concentration of cisplatin or doxorubicin are defined as medium control group. Cell viability was then determined by MTT assay. MTT in PBS was added into cells at a final concentration of 0.5 mg/ml. After 1 hour of incubation at 37°C, the medium was aspirated and 100 μl dimethylsulfoxide was added to dissolve the cells and the absorbance was measured at 570 nm.
Statistical analysis
Data were expressed as the mean ± standard deviation. Difference between any two groups was determined by analysis of variance. P < 0.05 was considered statistically significant.
Discussion
The NF-κB pathway plays an important role in immune response, inflammation, and cancer development [
25]. As a recently indentified kinase in the NF-κB pathway, IKKε is upregulated in a great proportion of breast cancer cells as well as tumor specimens [
6]. Our findings support the hypothesis that IKKε plays an important role in the tumorigenesis of breast cancer.
IKKε plays an important role in cell transformation, and activation of the NF-κB pathway is involved in the IKKε-mediated transformation [
6]. The tumor suppressor CYLD is directly phosphorylated by IKKε at serine-418 to decrease its deubiquitinase activity, which is essential to the IKKε-induced transformation [
7]. Moreover, breast cancer cells Hs578T stably expressing IKKε K38A (kinase-inactive IKKε) showed dramatically low colony formation ability in soft agar compared with cells transfected with the control vector (pCDNA3-FLAG-IKKε) [
11]. Consistent with these observations, we found that silencing of IKKε with siRNA led to significant reduction in focus formation in both MCF-7 cells and SK-BR-3 cells (Figure
2).
Several lines of evidence implicate that NF-κB and NF-κB-related IKKs are involved in cell invasion and tumor metastasis [
26,
27]. For example, prevention of IKKα activation resulted in inhibition of prostate cancer metastasis in TRAMP mice [
28]. For the first time, we conducted numerous experiments including the would-healing assay, migration assay, and invasion assay to assess the effect of IKKε siRNA on invasiveness properties of breast cancer cells. As shown in Figures
3 and
4, the invasiveness properties were significantly inhibited in cells treated with the IKKε siRNA in comparison with cells treated with the scrambled siRNA. These data are consistent with a previous report that breast cancer cells (NF639) transfected with IKKε K38A (kinase-inactive) vectors induced a less invasive phenotype compared with cells transfected with vectors expressing the active IKKε [
11].
Recent studies have shown that IKKε knockdown with lentiviral shRNA inhibited the proliferation and survival of transformed breast cancer cells, but not the nontransformed human mammary epithelial cells (MCF-10A) [
6]. A similar inhibition effect on cell proliferation was also observed in IKKε knockdown Hela cells and ovarian cancer cells [
4,
23]. In agreement with these findings, we observed a significant anti-proliferation effect of IKKε siRNA in breast cancer cells (Figure
5). To further elucidate the mechanism of this anti-proliferation effect, cell cycle analysis was conducted. A significant cell cycle arrest in the G
0/G
1 phase was observed (Figure
6). All these data strongly suggest the role of IKKε in breast cancer proliferation.
We next examined the effect of IKKε on cell apoptosis. There is some controversy regarding the role of IKKε in cell apoptosis. It has been reported that IKKε inhibition induces apoptosis in Hela cells [
29]. Another report using lentiviral shRNA targeting IKKε, however, did not show any apoptosis in ovarian cancer cells (A2780). Instead, overexpression of IKKε was found associated with cisplatin resistance. Significant apoptosis was detected in IKKε knockdown A2780 cells after 20 hours of exposure to cisplatin in comparison with cells treated with cisplatin alone [
23]. In the current study, we did not observe significant apoptosis in IKKε knockdown SK-BR-3 and MCF-7 cells after silencing IKKε using siRNA.
Although the relationship between IKKε and NF-κB is not fully understood, it was postulated that a significant fraction of NF-κB activation was induced by aberrant IKKε expression in tumor cells [
4,
6,
7]. Using the NF-κB transcriptional activity assay, we showed a significant reduction in basal NF-κB activity after IKKε suppression (Figure
8). This result is in agreement with a previous finding that IKKε knockdown in Hela cells reduced constitutive activity of the NF-κB dependent promoter 3X-κB [
4]. The correlation of IKKε with NF-κB may explain the role of IKKε in malignant transformation and invasiveness of tumor cells.
Moreover, we examined the expression of Bcl-2 and cyclin D
1, which are two important proteins regulated by the NF-κB pathway. Bcl-2 is an important apoptosis regulator involved in processing multiple death signals that are associated with mitochondria [
30]. The Bcl-2 expression level correlates with chemotherapy resistance [
31‐
33]. Downregulation of Bcl-2 results in induction of apoptosis and increased sensitivity to chemotherapy drugs [
34,
35]. Knockdown of Bcl-2 in MCF-7 cells using siRNA, however, only increased apoptosis by 9% (at 72 hours) and 11% (at 96 hours) in comparison with the control group [
36]. In addition, Akar and colleagues demonstrated that cell death (MCF-7 cells) triggered by Bcl-2 siRNA was caused by the induction of autophagic cell death rather than apoptosis. The authors did not observe any apoptosis effect in breast cancer cells upon Bcl-2 silencing [
37]. These controversial reports suggested that downregulation of the anti-apoptosis protein Bcl-2 alone does not necessarily result in apoptosis, especially considering the fact that induction of apoptosis is determined by a balance of multiple pro-apoptosis proteins and anti-apoptosis proteins [
38]. Similar to these findings, we only observed negligible apoptosis in breast cancer cells (Figure
7), although the Bcl-2 level was downregulated by the IKKε siRNA (Figure
9a to
9c). These results might be explained by a compensation of other existed anti-apoptosis factors. In addition, the treatment of IKKε siRNA did not sensitize breast cancer cells to cisplatin and doxorubicin (Figure
10), indicating that silencing IKKε alone may not be sufficient to induce cell apoptosis.
On the other hand, significant inhibition of cyclin D
1 was observed in cells treatment with IKKε siRNA (Figure
9d). Cyclin D
1, regulated by the NF-κB pathway, is overexpressed in more than 50% of breast cancers, and is identified as one of the most commonly upregulated proteins in breast cancer [
39,
40]. There is mounting evidence that cyclin D
1 plays a critical role in breast cancer cell cycle control. The induction of cyclin D
1 in breast cancer cells shortens the G
1 phase and increases the number of cells that progress through the G
1 phase, resulting in an increased proliferation [
21]. It was reported that overexpression of an inactive mutant of IKKε (K38A) in Hs578T cells resulted in reduction of cyclin D
1 [
11]. A recent study showed that IKKε phosphorylates estrogen receptor α at serine-167 and subsequently transcriptionally upregulates cyclin D
1 [
41]. Our results showed that cyclin D
1 expressions were downregulated upon IKKε knockdown in both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (SK-BR-3) breast cancer cells (Figure
9c, d), and the reduced cyclin D
1 expressions in both breast cancer cell lines were correlated with a cell cycle arrest in G
0/G
1 (Figure
6a, b).
Competing interests
The authors declare that they have a financial competing interest. The authors have submitted a patent disclosure relating to the content of this manuscript.
Authors' contributions
KC and BQ designed the research. BQ performed the research. KC and BQ analyzed the data. KC and BQ wrote the paper. All authors read and approved the final manuscript.