Cell culture and transfection
The MCF10ATw cell line was generated by stable transfection [58
] of pcDNA3-TWIST [59
] into MCF10A cells (an immortalized normal human breast epithelial cell line, American Type Culture Collection, ATCC, Manassas, VA, USA). MCF10ATw and MCF10A cells were cultured as previously described [60
]. HEK293, SKBR3, MCF7 and BT549 cell lines were purchased from ATCC and cultured using ATCC's recommended protocols and authentication methods. All cells were maintained at 37°C, 5% CO2
, 90% humidity in a tissue culture (TC) incubator. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
Triplicate samples of MCF10A and MCF10ATw cells were used for expression profiling with the Affymetrix Human Genome U133 Plus 2.0 array, and data collection was performed in the Microarray Core Facility at City of Hope. RNA was extracted using TRIzol reagent (Invitrogen) and analyzed for integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). Double stranded cDNA was reverse transcribed using total RNA (5 μg), the GeneChip® Expression 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA) and oligo-dT primers containing a T7 RNA polymerase promoter. A 1:10 dilution of Poly-A controls (2 μl) was added as an internal control for the synthesis. Double-stranded cDNA was used as a template to generate biotinylated cRNA using the GeneChip® Expression 3'-Amplification Reagents for in vitro transcription labeling (Affymetrix). Biotin-labeled cRNA was fragmented following the Affymetrix protocol. Hybridization cocktails contained 15 μg fragmented cRNA, 5 μl 3 nM control oligonucleotide B2, 15 μl 20X eukaryotic hybridization controls, BSA (0.5 mg/ml), herring sperm DNA (0.1 mg/ml), dimethyl sulfoxide (DMSO) (10%), and hybridization buffer for a final volume of 300 μl. Hybridization cocktails (200 μl) were hybridized (45°C, 16 h) to HG U133 Plus 2.0 Affymetrix arrays in an Affymetrix GeneChip Hybridization Oven 640. GeneChip arrays were washed with wash buffer (Affymetrix, santa Clara, CA, USA) and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 450, followed by scanning on an Affymetrix GeneArray 3000 scanner. Data were extracted using GeneChip Operating Software (version 1.4) (Affymetrix, Santa Clara, CA USA). Analysis of microarray data was performed using Partek Genomics Suite 6.4 (Partek, Inc., St. Louis, MO, USA) as follows. The Robust Multi-array Average (RMA) algorithm was adapted to normalize and summarize the intensities of probes into gene-level expression. A two-way ANOVA model was used with TWIST over-expression and scan dates as factors to identify the effect contributed mainly by TWIST. Only genes with P-value < 0.05 and |fold change| >2 were considered significantly differentially expressed. Genes were further analyzed using Gene Set enrichment Analysis (GSEA) to provide gene enrichment analysis and functional interpretation. A cytokine heat map was generated based on cytokine-related gene sets available from GSEA.
Conditioned media from cell cultures with equal numbers of seeded cells (8 × 105, six-well plate) cultured for 24 h were collected for ELISA (eBiosciences, San Diego, CA, USA).
Cytokine arrays, immunoblotting, and co-immunoprecipitations
Cytokine arrays (RayBiotech, Inc., Norcross, GA, USA) were blotted using the manufacturer's instructions. For immunoblotting, proteins were resolved by SDS-PAGE and probed with anti-E-Cad (Cell Signaling, Danvers, MA, USA), anti-N-Cad (Abcam, San Francisco, CA, USA), anti-Vimentin (R&D Systems, Minneapolis, MN, USA), anti-TWIST1 (Abcam), anti-RELA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Myc (EMD, San Diego, CA, USA) and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. For immunoprecipitation, HEK293 cells were solubilized in RIPA buffer, the lysate centrifuged (10,000 × g, 10 minutes) and the supernatant containing the soluble proteins collected. Protein lysates (100 μg) were first pre-cleared with normal IgG and protein A/G plus conjugated agarose beads (Santa Cruz Biotechnology, Inc.), then incubated with new beads and antibodies of interest overnight (4°C, on a rocker). Beads were washed five times (RIPA buffer) and boiled in Laemmli loading buffer in the presence of Dithiothreitol for further analysis. BT549 cells collected for fractionation-coupled co-IP were first resuspended in KCl (10 mM) hypotonic buffer and lysed with 0.4% IGEPAL (NP-40 substitute); nuclei were collected by centrifugation and solubilized with high salt buffer (0.4 M NaCl) by rocking (4°C, 1 h). Nuclear lysates were cleared by centrifugation (16,000 × g, 5 minutes) and the salt concentration was adjusted to 135 mM for co-IP experiments.
RT-PCR and quantitative PCR
Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). First strand cDNA was synthesized from 1 μg total RNA with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). PCR was performed for 35 cycles (95°C, 30 s; 57°C, 30 s; 72°C, 15 s) with Taq DNA polymerase (Invitrogen). Relative mRNA levels were quantified using SYBR supermix (Bio-Rad, Hercules, CA, USA) on an iCycler iQ5 for 40 cycles (95°C, 30 s; 57°C, 15 s; 72°C, 15 s) followed by default melting curve cycles and analyzed using IQ5 software by PCR baseline subtraction (Bio-Rad). The following primers were used: TWIST1 forward 5'-AGCAAGATTCAGACCCTCAAGC-3', reverse 5'-CTCCATCCTCCAGACCGAGA-3'; IL8 forward 5'-CTGTCTGGACCCCAAGGAAAACT-3', reverse 5'-GCAACCCTACAACAGACCCACAC-3'; β-ACTIN forward: 5'-CCGCAAAGACCTGTACGCCAAC-3', reverse 5'-CCAGGGCAGTGATCTCCTTCTG-3'.
Plasmids, shRNA constructs and viral production
The human IL8 promoter (bases -262 to -55) was amplified from MCF10A genomic DNA (DNeasy®
tissue kit, Qiagen) and cloned into pGL3 plasmid. pGL3-IL8 ΔκB and ΔE-box were generated by PCR site-directed mutagenesis (SDT). The coding sequences of TWIST1 full length and truncated (missing the last 20 amino acids, ΔWR) were amplified from pcDNA3-Tw [59
] and subcloned into pcDNA4. SDT were induced to generate the C432A A433G mutations (S144R K145E) and C352T mutation (R118C). WT pcDNA4-TWIST1 was subcloned into pENTR4 (Invitrogen) to shuttle into pAd/CMV/V5-DEST (Invitrogen) by LR clonase II (Invitrogen). Adenoviral particles were packaged using HEK293A cells, according to the manufacturer's guidelines, and titrated. The mRNA target sequences of shRNAs shTw1 (shTw) and shTw2 were 5'-GGACAAGCUGAGCAAGAUU-3' and 5'-GCGACGAGCUGGACTCCAA-3', respectively. The sequences of shCtrl (mRNA target sequence UUCUCCGAACGUGUCACGU) and shIL8 (mRNA target sequence GCCAAGGAGUGCUAAAGAA) were previously reported [61
]. shRNAs were created with siRNA sequences connected to a loop and a complementary sequence that were cloned into pcDNA3-U6 (gift from Dr. John Rossi, Beckman Research Institute of City of Hope). The U6-shRNA fragments were subcloned into pENTR4 (Invitrogen) to shuttle into pLenti6/Block-It™-DEST (Invitrogen). Lentiviral particles were generated, according to the manufacturer's guidelines, using HEK293FT cells. pGL3-3X κB, pCMV-IκBSR, pPCR-shGFP and pPCR-shRelA plasmids were gifts from Dr. Rama Natarajan (Beckman Research Institute of City of Hope).
Cells were seeded in 24-well plates and co-transfected with pGL3 firefly luciferase promoter construct, pSV40-Renilla luciferase (Promega, San Luis Obispo, CA, USA) and transcription factor constructs of interest. After transfection (24 h), cell lysates were collected and firefly/renilla luciferase activities were assayed for luminescence using the Dual-Luciferase Reporter Assay System (Promega).
Chromatin immunoprecipitation assay
ChIP assays were performed using the fast ChIP protocol [62
] with minor modifications as follows. Cells were cross-linked on a dish with 1.1% formaldehyde (10 minutes, room temperature), quenched with 1.25 M glycine solution (5 minutes, room temperature), and collected in 1 ml PBS with protease inhibitors cocktail (Roche Applied Science Indianapolis, IN, USA)). Cells were then processed and IPs performed as described [62
]. Precipitated chromatin was quantified using quantitative PCR and presented as percent of input. Anti-TWIST1 antibodies used for ChIP were custom-made to target the sequence GCQPPSGKRGGRKRRTSRRT. Anti-RELA antibodies were from Santa Cruz Biotechnology, Inc.; normal rabbit IgG was from Millipore (Temecula, CA, USA). Enrichment of IL8 promoter was analyzed by qPCR and presented by percent input using primers forward 5'-GTGATGACTCAGGTTTGCCC-3' and reverse 5'-GGTTGGTTTCTTCCTGGCTCT-3'. Control primers, forward 5'-ATCAGTCAAGCCAGGTTGTGTC-3', reverse 5'-AACACAGTGCATGGAGTGACAA-3', target a region 2 kb upstream of the transcription initiation site for the IL8 gene.
Transwell migration/invasion assays
Transwell inserts (Millipore, 8 μm pore diameter) were pre-coated with 1 mg/ml fibronectin and equilibrated with serum free medium in a TC incubator for 1 h Cells (3.75 × 105 for MCF10A and MCF10ATw cells, 1 × 105 for BT549 cells) were resuspended in culture medium supplemented with 0.25% serum (400 μl) and loaded onto the upper well of inserts. Media (600 μl) that contained 20% serum and indicated components of interest were added to the lower well. For invasion assays, Matrigel (60 μl, 3 mg/ml, diluted with serum-free medium, BD Biosciences San Jose, California, USA) was layered on the upper membrane and placed in the TC incubator for 30 minutes to solidify. Cells were allowed to migrate/invade for 24 h, followed by fixation with 4% paraformaldehye and staining with hematoxilin and eosin. Transwell membranes were removed and cells on the upper side cleaned off with a cotton tip. Membranes were then mounted and images taken of the upper, lower, left, right and center of the membrane. Migrated or invaded cells were quantified using Image-ProPlus5.1 (Media Cybernetics, Inc. Rockville, MD 20850 USA). and data presented as a sum of the five images. Neutralizing anti-IL8 antibodies were purchased from R&D Systems; IL8 inhibitors repertaxin and SB225002 were from Sigma.
Cells (3.75 × 104 for MCF10A and MCF10ATw cells; 104 for BT549 cells) were seeded in 96-well plates in equivalent conditions as for migration/invasion assays, and cell counts were determined with CellTiter 96 Aqueous One solution (Promega) according to the manufacturer's guidelines.
Conditioned media collected from MCF10A and MCF0ATw cells were filtered through a PVDF 0.45 μm low protein binding filter, concentrated using a 3000 NMWL Centricon concentrator (Millipore), mixed with β-mercaptoethanol-free loading buffer and resolved on a non-reducing PAGE gel that contained 0.1% (1 mg/ml) gelatin (Sigma). The gel was then incubated (1 h, room temperature, with shaking) in 2.5% Triton-X100 solution in water, followed by incubation (overnight, 37°C) in digestion buffer (10 mM calcium chloride, 20 mM Tris Acetic Acid, pH of 7.5). The gelatin gel was then stained with Coomassie Brilliant Blue (Bio-Rad) for 30 minutes at room temperature, washed with destaining solution (50:10:40 methanol:acetic acid:water) and imaged using a Kodak Electrophoresis Documentation and Analysis System 290 Eastman Kodak Company, Molecular Imaging Systems, New York, USA.
Bioinformatics and statistical analysis
STOCKHOLM (GSE1456) and UPPSALA (GSE3494) microarray data sets were downloaded from the NCBI Gene Expression Omnibus. The data were divided into subtypes [33
] based on information provided by Dr. Yudi Pawitan (Karolinska Institut, Sweden) and relative expression levels were represented by raw data. Non-parametric Mann-Whitney U test was performed to calculate the P