Cell culture and establishment of tamoxifen-resistant MCF-7 (MCF-7/TamR) cells
The human epithelial breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured under a humidified condition at 37 °C, 5% CO2 in RPMI 1640 medium (Gibco BRL, Carlsbad, CA, USA) containing 10% fetal bovine serum (Capricorn, Germany) and 2% penicillin/streptomycin (Capricorn). All cells were used within 12 passages after resuscitation of stocks. The MCF-7/TamR cells were generated by culturing MCF-7 cells in the presence of 4-hydroxytamoxifen (Tam) (Sigma-Aldrich, St. Louis, MO, USA) in complete RPMI 1640 medium. The cells were continuously exposed to increasing concentrations of Tam up to 160 nM over a period of 22 weeks, during which the medium was changed twice a week.
Study subjects
Solid tissues and slide-mounted formalin-fixed paraffin-embedded (FFPE) tissue sections of tumor samples were obtained from patients who underwent surgery between 2012 and 2013 at the National Cancer Center (NCC) in Korea. TamS tissues were obtained from patients who showed a clinical response to Tam, i.e., no tumor recurrence (n = 33). TamR tissues were obtained from patients who subsequently developed TamR (defined as disease recurrence while administering Tam; n = 28). Clinical details are presented in Table S1. All patients provided written informed consent to donate the removed tissues to NCC in Korea, and samples were obtained according to the protocols approved by the Research Ethics Board of NCC.
Generation of stable cell lines
Lentiviral particles with control clones and human ELOVL2 ORF clones containing C-terminal mGFP tag were purchased from OriGene (Rockville, MD, USA). MCF-7 and MCF-7/TamR cells were seeded at a density of 5 × 103 cells/well in a 96-well plate 1 day before transduction. The next day, the cells were infected with lentivirus for 4 h in the presence of 8 µg/mL polybrene (Sigma-Aldrich), and then the medium was replaced with a fresh complete medium. After 72 h, the cells were selected using 1 µg/mL puromycin (Thermo Fisher Scientific, Waltham, MA, USA) for 10 days.
Cell Transfection
siRNAs against ELOVL2 and THEM4 were purchased from Bioneer (Daejeon, Korea), and an ELOVL2-overexpressing vector was developed using the pEZ-MT02 plasmid vector (GeneCopoeia, Rockville, MD, USA) by CosmoGenetech (Seoul, Korea). All siRNAs were diluted in Opti-MEM Medium (Gibco BRL) with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), and the mixture was incubated for 5 min. The cells were transiently transfected at final concentrations of 20 nM or 40 nM with siRNA following the manufacturer’s instructions. Overexpression vectors (2 µg) were transfected into the cells using Lipofectamine 3000 transfection reagent (Invitrogen). After 24 h of transfection, cells were harvested and used for the following experiments. All results for the optimization of transfection are demonstrated in Fig. S1.
Cell proliferation assay
The cell growth rate was monitored by colony formation assay and colorimetric assay using CCK-8 reagent (Dojindo, Kumamoto, Japan). In all, 3 × 103 cells/well were seeded onto a 96-well plate and cultured up to 5–7 days. Following staining with CCK-8 solution according to the provided instructions, optical densities were measured on a microplate reader (Sunrise, Tecan, Switzerland) and OD595 was eliminated from the OD450. For colony formation assay, cells were seeded at a density of 3 × 103 cells/dish on a 60-mm culture dish. After transfection and Tam treatment, cells were maintained in a 5% CO2 incubator (37 °C) for 14–20 days. Colonies were fixed with a 7:1 mixture of methanol and acetic acid, stained using 0.2% crystal violet (Gibco BRL), and counted with ImageJ software (NIH, MD, USA).
Flow cytometric analysis
Apoptosis was analyzed using an APC Annexin V Apoptosis Detection Kit with PI (BioLegend, San Diego, CA, USA). Annexin V staining was performed for cells diluted in Annexin V binding buffer for 8 min followed by propidium iodide (PI) reagent treatment for 10 min. Samples were measured using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) with 488-nm and 640-nm lasers. To monitor Tam uptake by cells, 1 × 106 cells seeded in a 60-mm dish were treated with FLTX1 (Aobious, Gloucester, MA, USA, AOB4054) for 2 h at final concentration of 10 µM. Cells were then harvested after washing with PBS, and a concentration of 1 × 106 cells/mL was prepared. FLTX1 fluorescence was detected with Becton Dickinson FACSAria III (BD Biosciences) and analyzed with the Flowing Software 2.5 (http://flowingsoftware.btk.fi/).
Tam sensitivity assay
Alterations in sensitivity to Tam were measured by cytotoxicity assay. Briefly, 1 × 104 cells were seeded per well in a 96-well plate and transfected with recombinant cDNA-harboring plasmids and/or siRNAs. On the following day, Tam dissolved in sterile-filtered ethanol was added to cells at final concentrations of 0, 0.05, 0.1, 0.5, and 2 µM with a final ethanol concentration 0.1%. After 24 h, 10 µL of CCK-8 solution was added to each well and the plate was incubated for 90 min. Following this, the plate was read at 450 nm on a plate reader.
Methylation and expression microarray experiment
Genome-wide methylation analysis was performed with Macrogen (Seoul, Korea) on Illumina Infinium Human Methylation 450K and Illumina Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA, USA) covering over 450,000 and 850,000 CpG sites, respectively, to compare the DNA methylation profiles between MCF-7 and MCF-7/TamR. All arrays were processed with Illumina GenomeStudio v2011.1. To identify global gene expression profiles, total RNA of MCF-7/TamR/ORF NC or MCF-7/TamR/ELOVL2 ORF cells was profiled using the SurePrint G3 Human Gene Expression 8 × 60K v3 microarray technology (Agilent, Santa Clara, CA, USA) containing 58,201 probes by Lugen Sci (Seoul, Korea). Agilent Feature Extraction software (v11.0.1.1) was used to extract and process raw data. The microarray data are deposited in the GEO database website (http://www.ncbi.nlm.nih.gov/geo/) with the SuperSeries accession number GSE132617: expression array, GSE132614; methylation array, GSE132615 and GSE132616.
Pathway and clustering analysis
Ingenuity Pathway Analysis tool (Ingenuity Systems, Redwood City, CA, USA) was used to generate significant networks and biological functions for the differentially methylated genes within the promoter (|Δβ| ≥ 0.2, P-value < 0.05) by acquisition of Tam resistance, and for the differentially expressed genes (|fold change| ≥ 2, P-value < 0.05) by overexpressing ELOVL2 in MCF-7/TamR. Genes with significant changes were clustered using Clustering 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/) and the results were visualized using the TreeView v1.1.6 program (http://jtreeview. sourceforge.net/).
Methylation-specific PCR (MSP) and quantitative real-time RT-PCR (qPCR)
MSP was performed to determine the methylation level of specific CpG sites, as previously described [20]. Briefly, DNA and RNA from FFPE sections were extracted using the RecoverAll Multi-Sample RNA/DNA Workflow (Invitrogen). Total DNA and RNA were prepared using ZR-Duet DNA/RNA MiniPrep kit (Zymo research, Irvine, CA, USA) from solid tissues and cultured cells. For preparing samples for methylation analysis, the genomic DNA was treated with bisulfite using a Zymo Research EZ DNA Methylation Kit (Zymo Research). Demethylation of the cytosine residues was achieved by exposing the cells to culture media containing a methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (Aza) (Sigma-Aldrich), at a concentration of 5 µM for 72 h. PCR was conducted using 4–8 ng of DNA, and the yielded signals were calculated. To identify the transcript level of coding genes, cDNA was synthesized using a ReverTra Ace qPCR RT MasterMix with gDNA Remover kit (Toyobo, Osaka, Japan). qPCR analysis was conducted using KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) on an ABI 7300 instrument (Applied Biosystems, Foster City, CA, USA). Oligonucleotide primers were purchased from Bionics (Daejeon, Korea) (Table S2).
Western blot analysis
Protein extraction from cultured cells and Western blot analysis were performed as previously described [21]. The following antibodies were used: anti-ELOVL2 (1:500, Bioss, Woburn, MA, USA, bs-7053R), anti-THEM4 (1:500, Abcam, Cambridge, MA, USA, ab106435), anti-β-Actin (1:1000, Bioss, bs-0061R), anti-phospho-AKT (1:300, Bioss, bs-5182R), anti-AKT (1:2500, Abcam, ab179463), and HRP-conjugated anti-rabbit IgG antibody (1:1000, GeneTex, Irvine, CA, USA, GTX213110-01). The bands on the membrane were detected using the ECL reagent (Abfrontier, Seoul, Korea) and analyzed with Image Lab software (Bio-Rad, Herculer, CA, USA). The whole blot can be accessed in Fig. S2.
Tumor xenograft experiments
All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Dongguk University (No: IACUC-2017-010-1). We used 6- to 7-week-old female BALB/c nude mice (Orient Bio, Seongnam, Korea) for MCF-7 and MCF-7/TamR-derived xenograft models. The mice were anesthetized with a mixture of isoflurane (Piramal Critical Care, Mettawa, IL, USA) and oxygen, and administered 17β-estradiol pellets (0.72 mg/pellet total dose; Innovative Research of America, Sonnasota, FL, USA) subcutaneously in the lateral neck area. On the next day, subcutaneous injections of 1 × 107 breast cancer cells resuspended with 100 µL of 1:1 mixture of PBS (Gibco BRL) and Matrigel (BD Biosciences, Bedford, MA, USA) were administered to the mice. Tumor growth was monitored weekly, and tumor volumes were calculated based on the following formula: length × width2 × 0.5. When tumor sizes reached approximately 100 mm3, the mice were randomized into two groups for Tam treatment (Sigma-Aldrich). One group received intraperitoneal administration of 100 µL of 1 mg/kg Tam in corn oil (Sigma-Aldrich) and the other group was injected with a vehicle control for 5 days a week during the experiment. After 7 weeks of implantation, animals were sacrificed and tumors were harvested. The cancer tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks for histological analysis by Logone Bio (Seoul, Korea).
Immunohistochemical staining
Immunohistochemical analysis was performed using tumor tissues of xenograft mice. To do this, paraffin blocks were sectioned 10 µm thick, organized into slides, and rehydrated through a graded ethanol series. Endogenous peroxidase activity in sections was ceased with 0.3% H2O2 treatment for 15 min and then rabbit anti-ELOVL2 (1:400, Bioss, bs-7053R) or rabbit anti-THEM4 (1:100, Abcam, ab106435) was applied for 1 h at room temperature followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibodies (Dako, Glostrup, Denmark, K4003). Liquid diaminobenzidine tetrahydrochloride (DAB) (Dako, K3468) was used as a chromogen to detect horseradish peroxidase activity. After counterstaining with Mayer's hematoxylin, immunohistochemical images were generated using panoramic MIDI scanner (3Dhistech, Budapest, Hungary). The ImageJ program (NIH) was used to profile the DAB-positive areas of immunohistochemical images.
Statistical analysis
For microarray data, observations with adjusted P-values ≥ 0.05 were removed and were excluded from further analysis. Adjustments were made to control for false discoveries. Following adjustments, the remaining genes were defined as differentially methylated if they displayed an increased or decreased methylation level which was equal to or higher than 0.2 compared with the control, or differentially expressed if they displayed at least a 2-fold difference compared with the control. Student’s t-test was implemented to demonstrate statistical significance for all data from qPCR, MSP, IHC, and Western blot analysis comparing samples and control groups. Chi-squared test was used to analyze the differences in the rate of each variable for tumor tissues. Statistical analyses were conducted using SPSS for Windows, release 17.0 (SPSS Inc., Chicago, IL, USA). The results are expressed as the mean ± standard error and considered statistically significant at P-value < 0.05.