Materials
YC-1, PD98059, LY 294002, and H-89, and PP2 were purchased from Sigma Aldrich Chemical Co. (St. Louis, Mo, USA). Penicillin–streptomycin solution, fetal bovine serum (FBS), trypsin, TurboFect, and Lipofectamine 2000 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The pCMV-b-gal plasmid was obtained from Clontech (Palo Alto, CA, USA). The Dual-Glo luciferase assay kit, pERE-Luc plasmid, and the mammalian-two-hybrid system were obtained from Promega (Madison, WI, USA). Antibodies against PKA/p-PKA, Akt/p-Akt, ERK/p-ERK, SRC/p-SRC, p-ERα, and horseradish peroxidase-linked anti-mouse and rabbit IgG were obtained from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against β-actin and CYP1B1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The HIF-1α antibody was obtained from Abnova (Taipei City, Taiwan), and the ERα antibody was obtained from Abcam (Cambridge, UK). The antibody against p-ERa (Ser305) was obtained from Bethyl Laboratories (Montgomery, Texas, USA). PE-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Oligonucleotide primers for polymerase chain reaction (PCR) were custom synthesized by Bioneer (South Korea). All chemicals used were of the highest commercially available grade.
Cell culture and treatment
The human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) were obtained from the Korea Cell Line Bank (KCLB, Seoul, South Korea) and the normal breast epithelial cell line MCF10A was obtained from the Laboratory Animal Resource Center (Korea Research Institute of Bioscience and Biotechnology, South Korea). MCF-7 and MDA-MB-231 cells were cultured in DMEM from HyClone (Thermo Fisher Scientific) supplemented with 10% FBS in a humidified 5% CO2 incubator at 37 °C. MCF10A cells were cultured in Human Mammary Epithelial Cell Systems (HMEM) BulletKit medium from Lonza (Basel, Switzerland) and only cells up to passage 15 were used in the experiments. To induce hypoxia, these cells were cultured in a hypoxia chamber (Billups-Rothenberg, San Diego, CA, USA) containing a 1% O2 gas mixture. Cell lines were DNA fingerprinted by the Korea Cell Line Bank (KCLB, Seoul, South Korea). The stock solution of the inhibitor was diluted with dimethylsulfoxide (DMSO) and added directly to the culture medium. Control cells were treated with DMSO only, and the final DMSO concentration was always <0.2%.
Immunoblot
Protein lysates were prepared from cells cultured under hypoxia or normoxic conditions, and proteins were quantified using the Bradford protein assay (Bio-Rad, Irvine, CA, USA). The lysates were electroblotted on polyvinylidene difluoride membranes and reacted with the primary antibodies and secondary antibodies. The membranes were visualized with enhanced chemiluminescence (ECL) solution and analyzed using a ChemiDoc Imager (Bio-Rad).
Transient transfection and luciferase assays
Cells were transiently transfected with the human CYP1B1 promoter-luciferase (Luc) full length (FL), pERE-Luc, and pCMV-b-gal using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, MA, USA). The CYP1B1 promoter-Luc FL vector (−1635 to +1588) was gifted by Dr. Robert Barouki [27]. The CYP1B1-Luc FL and deletion plasmids (−910 to +25 and −91 to +25) were used to investigate the hypoxia-mediated CYP1B1 promoter binding sites. After transfection, cells were incubated for 24 h under normoxic or hypoxic conditions, and cell lysates for luciferase reporter assay were prepared. Cell lysates were mixed with an equal volume of luciferase assay substrate reagent and analyzed using a luminometer (SpectraMax M5; Molecular Devices).
Gene silencing using small interfering RNA (siRNA)
The expressions of HIF-1α and ERα were knocked down using siRNA, and the results were analyzed by real-time qPCR, chromatin immunoprecipitation, and immunoblot analysis. siRNAs targeting HIF-1A and ESR1 mRNA and non-targeting siRNAs were purchased from OriGene (OriGene Inc, Rockville, MD, USA). Cells were transiently transfected with HIF-1α--siRNA, ERα-siRNA, or scrambled control siRNA using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, MA, USA).
RNA extraction and digital PCR analysis
Under hypoxia or normoxia, total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) and reverse-transcribed using RNA-to-cDNA EcoDry reagent (TaKaRa, Japan). PCR products were directly monitored during PCR assays using QuantStudio 3 real-time PCR system (Applied Biosystems) and detected via SYBR Gene reporter dye enhancement. The levels of CYP1B1 and β-actin mRNA in MCF7 cells were compared with those in control cells using the comparative cycle threshold (Ct) method.
Chromatin immunoprecipitation (ChIP) assay
Cells cultured under normoxic or hypoxic conditions were treated with formaldehyde to cross-link protein and DNA. The cells were then sonicated and chromatin-DNA complexes were precipitated using an antibody against ERα or non-specific mouse IgG using an EZ CHIP kit (Upstate, Lake Placid, NY, USA). PCR was performed using purified DNA, oligonucleotide PCR primers, and Taq DNA polymerase (TaKaRa). PCR products were analyzed on an agarose gel using the SYBR Safe DNA Gel Stain kit.
Co-immunoprecipitation (Co-IP) assay
Cells were cultured under hypoxic or normoxic conditions and lysed with IP buffer (Roche). Protein lysates were pre-removed by incubation with Protein A beads (GE Healthcare) and then incubated with beads and ERα antibodies. Antibody-bound beads were washed with PBS, electrophoresed on SDS-PAGE, and then immunoblotted with HIF-1α.
Mammalian two-hybrid (M2H) assay
Vectors [pBIND-ERα 1-180 (N-terminal domain, NTD), 180-302 (DNA binding domain, DBD), 302-595 (ligand-binding domain, LBD), pACT-HIF-1α FL, and pG5-Luc] were used for the M2H assay. Transfection was performed using the TurboFect transfection reagent (Thermo Fisher Scientific). The cells were lysed using lysis buffer (Promega) and the lysates were added to luminescent white plates. Luciferase activity was measured using a luminometer (SpectraMax M5). Relative reporter assay activity was calculated using the pG5-Luc control vector.
Immunofluorescence analysis of tumor microarrays (TMAs)
A human breast cancer tissue microarray (TMA, #BC081120e) containing 110 cases was purchased from Biomax (Rockville, MD). The immunofluorescent image was quantified using a quantitative analysis system (Thunder, Leica). Image analysis was performed using the LAS-X software (Leica) by acquiring fluorescence images of DAPI, CYP1B1, and HIF-1α for each TMA spot and scoring the sum of the target pixel intensities. Tumors were divided into high and low groups according to the ER expression score (according to the patient information sheet). The correlation of CYP1B1 and HIF-1α in ERα-positive breast tumors was assessed using linear (Pearson) and nonparametric (Spearman) correlation coefficients.
The cancer genome atlas (TCGA) data and cBioPortal
Ethical approval for the study was not required due to the retrospective nature of this study using only publicly available data. Transcriptome analysis data and pathological data of human breast cancer biopsy samples were obtained from the following dataset in cBioPortal platform: BRCA-TCGA-pub2015 [817 cases with mRNA data (RNA Seq V2 RSEM)], BRCA-TCGA [1100 cases], BRCA-METABRIC [1904 cases with mRNA data (microarray)]. Gene expression levels were normalized (Z-score) and scaled (log-transformed). Co-expression, overall survival, and volcano plots were calculated according to cBioPortal's online instructions and analyzed using the GraphPad Prism 9.00 software. The BRCA-METABRIC dataset was analyzed using the cBioPortal online tool for exploration and comparison between the two patient groups, and 304 genes differentially expressed in the ERα-positive high profile/HIF1α-CYP1B1 high group was filtered (q-value < 0.01, |Log2(FC)| > 0.58) and subjected to gene enrichment analysis using the Metascape platform (http://metascape.org) for gene annotation. The filtered data were subjected to hierarchical clustering and visualized as a heatmap.
Statistical analysis
Data are presented as ± standard error of the mean (SEM). All statistical analyses were performed using Student’s t-test or the Mann–Whitney U-test when data were not normally distributed. Statistical significance was set at P < 0.05.