Cell lines and culture conditions
Breast cancer cell lines MDA MB 231 (cat. No. HTB-26) [claudin-low TNBC], MDA MB 453 (cat. No. HTB-131) [HER2 (ER-,PR-, HER2+)], MDA MB 468 (cat. No. HTB-132) [basal TNBC], HCC-38 (CRL-2314) [claudin-low TNBC], BT-549 (cat. No. HTB-122) [basal TNBC], and BT-474 (cat. No. HTB-20) [luminal B (ER-, PR+,HER2+] were purchased from American Type Culture Collection (ATCC, Manassas, VA). Unless otherwise noted, all breast cancer cell lines were cultured in RPMI 1640 (Gibco, Thermo Fisher, Grand Island, NY) containing 10% FBS (Gemini, West Sacramento, CA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher, Grand Island, NY) and maintained at 37°C in a 5% CO2 atmosphere.
Expression and shRNA constructs
pBabeNeo (plasmid #1767) and pBabe-Neo-Flag-IKBKE (plasmid #15265) were purchased from Addgene. Transduced cells were cultured in the presence of 200μg/ml neomycin for 7 days. Use of IKBKE short hairpin (shRNA) constructs has been previously described . Two rounds of viral supernatants were applied to breast cancer cell lines over the course of 48 h, followed by incubation with growth medium for 24 h and selection with 2 μg/mL puromycin for 7 days. Selected transduced cells were used for all assays. Sequences of shRNA constructs: non-targeting control (shNeg):
Cells were cultured for 24 h to 50% confluence before transfection with Dharmacon On-Targetplus SMARTpool short interfering (siRNA) duplexes (NF-kB2, cat. No. L-003918-00; non-targeting control, cat. No. D-001810-10; IKBKE, cat. No. L-003723-00) according to manufacturer’s instructions (GE Dharmacon, Lafayette, CO). Briefly, cells were transfected with Dharmafect 1 transfection reagent (GE Dharmacon, Lafayette, CO) and individual siRNAs at a final concentration of 1% v/v and 25 nM, respectively. Cells were maintained in the presence of transfection reagent under normal culture conditions for 24 h before being used in assays.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) per manufacturer’s instructions and treated with DNAse. Final RNA concentration was determined using a NanoDrop spectrophotometer. RNA was reverse transcribed using Taqman reagents (Applied Biosystems) and gene expression was measured using Taqman probes on a ViiA7 Real-time PCR machine (Applied Biosystems). GAPDH was used as a control and quantitation of gene expression was accomplished using comparative threshold cycle ΔΔCT. Primers were purchased from Applied Biosystems (p52 cat. No. Hs01028901_g1, CXCL1 cat. No. Hs00236937_m1, CD44 cat. No. Hs01075861_m1, and GAPDH cat number: 4325792.
Whole cell protein was extracted from breast cancer cell lines using standard methods with NP-40 lysis buffer. Protein concentrations were determined using BCA Protein Assay Kit (Pierce, Thermo Scientific, Rockford, IL). SDS-PAGE was performed using the NuPage system (Invitrogen) and Luminata HRP Chemiluminescent Detection Reagents (Millipore, Temecula, CA). Antibodies were purchased from Sigma (IKKε, cat. No. I4907),
Abcam (IKKβ, cat. No. ab32135), Millipore (GAPDH, cat. No. MAB374; p100/52 cat. No. 05–361), Santa Cruz (p65, cat. No. sc-372), and Cell Signaling (IKKα, cat. No. 2682; pERK1/2, cat. No. 4377; Erk1/2, cat. No. 9102; phospho-p-65 (Ser536), cat. No. 3033).
Chromatin immunoprecipitation-qPCR (ChIP-qPCR) assay
The SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads) was purchased from Cell Signaling Technology (Danvers, MA). Assays were performed according to the manufacturer’s instructions. The antibody for p52 was purchased from Santa Cruz Biotechnology (cat. No. sc-7386 X). Promo was used to evaluate DNA sequences for transcription factor binding sites (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). The first 5000 bases upstream of the transcription start site were screened for binding motifs that correspond to NF-κB consensus binding sequence. The quantification of transcription factor binding to target genes was calculated by measuring the ratio of ChIP-to-Input and normal rabbit IgG antibody served as a negative control. Primer sequences for NF-kB binding sites on CXCL1 promoter: − 2.5 kb site: forward GATTTCCAGGCTCAAGGATGTA, reverse TCATTCAGTCTTCCAAACAAGC; − 02. Kb site: forward ATCCCAGAGTCTCAGAGTCCAC, reverse AAATTCCCGGAGTTCCAGAT.
Immunoprecipitation was performed on MDA MB 468 cells using the Abcam immunoprecipitation kit (cat. No. ab206996), according to manufacturer’s instructions. Briefly, non-denaturing lysis buffer was used to collect 300 μg of cell lysate was incubated overnight with 3 μg/ml of either control rabbit IgG (Santa Cruz, cat. No. sc-2027) or IKKε rabbit polyclonal antibody (Abcam, cat. No. ab7891). Antibody bound proteins were captured using protein A/G sepharose beads, eluted, and analyzed via western blot. Antibodies used for western blot detection were purchased from Sigma (IKKε, cat. No. I4907), Santa Cruz (IKKα, cat. No. sc-7606 and NIK cat. No. sc-8417).
MEK inhibitor, AZD6244, and a non-selective inhibitor of Ser/Thr kinases, BX795, that inhibits IKKε, among others were purchased from Selleck Chemicals (Houston, TX). The IKKβ inhibitor IKK-2 inhibitor IV from Calbiochem (San Diego, CA). The breast cancer cell growth was assessed using XTT as described . Cells were seeded in 96-well plates at a density of 2000 cells/50 μl/well. Plates were incubated for up to 9 days with medium and/or drug replenished every 3–4 days. Growth was assessed by incubating cultures with XTT for 3 h and absorbance read in a Tecan plate reader (Research Triangle Park, NC). Cell density in experimental wells was expressed as percent control. Experiments included triplicate samples and were repeated at least three times. IC50 values were calculated using CalcuSyn software (Paramus, NJ) and compared with publicly available database (www.cancerrxgene.org).
Nuclear lysates were extracted using a Nuclear Extraction Kit according to manufacturer’s instructions (Active Motif, Carlsbad, CA). Protein concentrations were determined using BCA Protein Assay Kit (Pierce, Thermo Scientific, Rockford, IL). NF-κB transcription factor binding was assessed using the TransAM NFκB Family ELISA Kit according to manufacturer’s instructions (Active Motif, Carlsbad, CA). 10 μg of nuclear extracts/20 μl/well were analyzed in triplicate and repeated at least three times.
Invasion potential of breast cancer cells was assessed using Cultrex 96-well BME Cell Invasion Assays (Trevigen, Gaithersburg, MD) according to manufacturer’s specifications. Briefly, 5 × 104 cells suspended in serum-free RPMI were plated in BME coated chambers, and allowed to migrate for 48 h using RPMI containing 10% FBS as a chemoattractant. Cells that migrated through BME chambers were stained with calcein, solubilized, and numbers assessed by measuring fluorescence in a Tecan fluorimeter (Tecan, Research Triangle Park, NC). Migrated cell numbers in triplicate samples were reported as percent control.
Anchorage-independent, low-attachment (LA), growth was evaluated using the CytoSelect 96-Well Anoikis Assay according to manufacturer’s protocol (Cell Biolabs, Inc., San Diego, CA). Briefly, 1 × 104 cells/100 μl/well were plated in quadruplicate on a 96-well anchorage resistant plate and in a companion standard culture plate with cell high attachment capability (HA) plate and cultured for 48 h. Anoikis was assessed by dual staining with calcein AM and ethidium homodimer followed by measurement of fluorescence in a Tecan fluorimeter (Tecan, Research Triangle Park, NC). Experiments were repeated at least three times. Fluorescence intensity was expressed relative to control treatment.
Spheroid formation assay
To generate breast cancer spheroids, 500 cells/well were cultured in serum free media containing EGF (20 ng/ml) and FGF (10 ng/ml) (Sigma-Aldrich, St. Louis, MO) in ultra-low attachment 96 well plates (Corning, Corning, NY). After 96 h, spheroids were imaged using AxioVision Rel. 4.8 through an Axio Observer A1 Inverted Microscope (Zeiss) and analyzed using ImageJ Software 1.48v . Spheroids with diameter ≥ 50 μm were quantified. Spheroid efficiency was calculated using the formula (spheroids ≥50 μm/ cells per well).
All statistical analysis was performed using Prism (GraphPad) using data acquired from at least three biological replicates. P < 0.05 was considered statistically significant. P values were calculated as described in figure legends. Error bars represent standard error of the mean.