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Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines
© Plummer et al; licensee BioMed Central Ltd. 2004
Received: 21 September 2004
Accepted: 16 December 2004
Published: 16 December 2004
Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling.
Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting.
Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells.
Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer.
Breast cancer is the leading cancer in women  and estrogen receptor (ER)(-) breast cancers have a poorer prognosis than ER(+) cancers [2, 3]. Smoking is a controversial risk factor for the development of these malignancies [4–7]. However, increases in pulmonary metastatic disease and lung cancer have been seen in smokers with breast cancer [8, 9]. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) causes cancer of the oral cavity, esophagus, respiratory tract and pancreas, but no breast cancer in laboratory animals  and has not been implicated in breast carcinogenesis to date.
Recent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control [11–15]. Studies in a cohort of 2442 men found an inverse association between risk of incident adenocarcinomas of the prostate and use of antihypertensive medication, including beta-blockers . The tobacco-specific carcinogenic nitrosamine NNK has recently been identified as a high affinity β-adrenergic agonist that stimulated the growth of pulmonary and pancreatic adenocarcinomas in vitro and in animal models [11, 13, 15]. The expression of β-adrenergic receptors has been correlated with the over-expression of the arachidonic acid-metabolizing enzymes cyclooxygenase-2 (COX-2) and lipoxygenases (LOX) in adenocarcinomas of lungs , colon , prostate , and pancreas . Inhibitors of these enzymes have been identified as cancer preventive agents in animal models of these cancers [13, 20–22]. Collectively, these findings suggest that among the superfamily of adenocarcinomas at various organ sites, there is a subset of malignancies that is regulated by β-adrenergic and arachidonic acid-mediated signal transduction pathways.
The majority of breast cancers are also adenocarcinomas and many of them over express COX-2 and/or LOX . This raises the possibility that comparable to findings in adenocarcinomas of the lungs, pancreas, colon and prostate, a subset of breast cancers may also be under beta-adrenergic control. In support of this hypothesis, studies have demonstrated that three estrogen-responsive and three non-estrogen responsive human cell lines derived from breast adenocarcinomas demonstrated a significant reduction in DNA synthesis in response to beta-blockers or inhibitors of the arachidonic acid-metabolizing enzymes COX-2 and 5-LOX . In addition, analysis by reverse transcription polymerase chain reaction (RT-PCR) revealed expression of β2-adrenergic receptors in all six breast cancer cell lines tested (MDA-MB-361, ZR-75-1, MCF-7, MDA-MB-453, MDA-MB-468, MDA-MB-435S), whereas β1 receptors were not found in two estrogen non-responsive cell lines (MDA-MB-435S, MDA-MB-453) .
Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers tested , and this expression of GIRK1 was associated with a more aggressive clinical behavior. Increases in GIRK currents by beta-adrenergic stimulation have been reported in adult rat cardiomyocytes and in Xenopus laevis oocytes coexpressing β2-adrenergic receptors and GIRK1/GIRK4 subunits . In addition, in rat atrial myocytes transiently transfected with β1 or β2 adrenergic receptors, the beta-adrenergic agonist isoproterenol stimulated GIRK currents, whereas this stimulation was not seen in non-transfected cells . The current investigations test the hypothesis that GIRK1 channels in human breast cancers are correlated with beta-adrenergic control.
The ER(+) human breast cancer cell lines MDA-MB-361, ZR-75-1, and MCF-7 and the ER(-) cell lines MDA-MB-453, MDA-MB-468 and MDA-MB-435S were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%, v/v), L-glutamine (2 mM), 100 U/ml of penicillin and 100 μg/ml streptomycin (Invitrogen-Life Technologies, Grand Island, NY) in an environment of 5% CO2. Exposure of cells to propranolol, isoproterenol, or clozapine (Sigma, St. Louis, MO), NNK (Chemsyn, Lexena, KS), or formoterol hemifumarate (Tocris, Ballwin, MO) for experiments was as detailed in the Figure Legends.
RNA was isolated by Trizol reagent (Invitrogen-Life Technologies) or by an Absolutely RNA kit (Stratagene, La Jolla, CA). RT-PCR was done as previously described . The GIRK1 primers are forward 5'-ctatggctaccgatacatcacag-3' and reverse 5'-ctgttcagtttgcatgcttcgc-3' which span exon 1 and 2  and amplifies a 441 bp fragment (bases 631–1072, Genbank Acession # NM_002239). The GIRK2 primers are forward 5'-atggatcaggacgtcgaaag-3' and reverse 5'-atctgtgatgacccggtagc-3' amplifies a 438 bp fragment (bases 700–1137, Genbank Acession #U52153). The GIRK4 primers are forward 5'-aaccaggacatggagattgg-3' and reverse 5'-gagaacaggaaagcggacac-3' which amplifies a 401 bp fragment (bases 117–517, Genbank Acession # L47208). PCR conditions are 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec for 40 cycles. Cyclophylin primers were used as an internal control (Ambion, Austin, TX).
Relative competitive RT-PCR
Preliminary experiments were done with MDA-MB-453 cells to determine a cycle number of PCR amplification that is within the linear range, which is critical for meaningful results to compare expression levels between samples and to determine the mixture of 18S primers/18S competimers (Ambion-Classic II). The 18S ribosomal RNA primers/competimers are used as an invariant internal control, which allows correction for sample variation. Results indicated this was 31 cycles of PCR and a 1:9 18S primer/competimers ratio. For experimental treatments, as described before , cDNA was made and PCR performed except reactions were spiked with 5 μCi [α-32P]-dCTP (3000 Ci/mmole, Dupont-NEN, Boston, MA). Reactions were run with the following conditions: 1 cycle of 2 min. at 94°C, then 31 cycles of 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec. A 10 μl sample of each PCR reaction was heated at 95°C for 3 min., then loaded into a 5% TBE-urea Ready Gel (Bio-Rad, Hercules, CA). This underwent electrophoresis at 200 V in TBE buffer until the xylene cyanol dye front reached the bottom of the gel. The gel was transferred to filter paper, dried and exposed to film or imaged on a Molecular Dynamics 445 SI phosphoimager (Sunnyvale, CA). A 100 bp DNA ladder (Invitrogen-Life Technologies) was exchange labeled with T4 polynucleotide kinase and 30 μCi [γ-32P] ATP (3000 Ci/mM, Dupont-NEN).
The GIRK-1 primers for real time PCR are forward 5'-ctctcggacctcttcaccac-3' and reverse 5'-gccacggtgtaggtgagaat-3' (bases 398–477, Genbank Acession # NM002239). and the internal TaqMan probe is 6-FAM-tcaagtggcgctggaacctc-TAMRA (bases 429–449, Sigma-Genosys, The Woodlands, TX), annealing temperature 62°. GIRK2 primers-forward 5'-gacctgccaagacacatcag-3' and reverse 5'-cggtcaggtagcgataggtc-3' (bases 766–886, Genbank Acession # U52153) and the internal TaqMan probe is 6-FAM-gtgcaatgttcatcacggcaac-TAMRA (bases 837–859), annealing temperature 56°. GIRK4 primers-forward 5'-agcgctacatggagaagagc-3' and reverse 5'-aagttgaagcgccacttgag-3' (bases 241–358, Genbank Acession # L47208) and the internal TaqMan probe is 6-FAM-accggtacctgagtgacctcttca-TAMRA (bases 301–324), annealing temperature 62°. Reactions were run on a Cepheid SmartCycler (Sunnyvale, CA). Reaction conditions are 200 μM dNTPs, 0.3 μM gene specific primers, 0.2 μM TaqMan probe, 4 mM (GIRK1) or 6 mM (GIRK2or4) magnesium acetate, 2 μl cDNA and 1.5 U MasterTaq (Eppendorf, Westbury, NY) and MasterTaq buffer in a final volume of 25 μl. TaqMan beta-actin detection reagents (Applied Biosystems) were used with the same reaction conditions as above except a 5 mM magnesium concentration was used and this was run at 95° for 120 seconds, followed by 45 cycles of 95°, 15 seconds; 68°, 30 seconds.
Measurement of potassium flux
We determined inward potassium flux in these cells by flow cytometry via the method of Krjukova et al. . The negatively charged fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC4(3)) (Molecular Probes, Eugene, OR) was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 106 cells at a final concentration of 150 × 10-9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter (San Jose, CA).
Analysis of protein expression by western blots
Following incubation with agents as detailed in the Figure legends, cells were washed twice with phosphate buffered saline and lysed with cold RIPA lysis buffer containing protease inhibitors (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1% Triton × 100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 0.5 mM DTT). Cell lysates were collected from culture plates using a rubber policeman, and protein collected by centrifugation. Protein concentrations were determined by BCA protein assay (Pierce, Rockford, IL). Aliquots of 20 μg protein were boiled in 2x loading buffer (0.1 M Tris-Cl, pH 6.8, 4% SDS, 0.2% Bromophenyl blue, 20% glycerol) for 4 minutes, then loaded onto 10% Tris-HCl-Polyacrylamide gels (Biorad, Hercules, CA), and transferred electrophoretically to nictrocellulose membranes. Membranes were incubated with primary antibodies (phospho-Erk; Cell Signaling, Beverly, MA) and appropriate secondary antibodies (Cell Signaling or Rockland, Gilbertsville, PA or Molecular Probes, Eugene OR). In all western blots, membranes were additionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The antibody-protein complexes were detected as previous described  or by the LiCor Odyssey infrared imaging system (Lincoln, NE).
Our data demonstrate expression of the G-protein inwardly rectifying potassium channel 1 (GIRK1) in 67% of the breast cancer cell lines tested, with higher levels in ER(+) cell lines. Approximately 40% of primary human breast cancers were found to express GIRK1 and expression of GIRK1 was not found to be correlated with ER status . These differences in our studies may be due to the subset of breast cancer cell lines tested. We also found that the normal breast epithelial cell line MCF 10A lacked GIRK1 expression (data not shown). GIRK1 cannot form functional channels by itself, other GIRK channels are needed . All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines.
The majority of experiments in the present study were done with the ER(-) cell line MDA-MB-453 since it was the only ER(-) cell line tested that expressed GIRK1, and because ER(-) breast cancers have a poorer prognosis than ER(+) cancers [2, 3]. We saw a significant increase in GIRK1 channel mRNA expression after 6 days of continuous exposure to propranolol in MDA-MB-453 cells. It is clear that at least six days of continuous exposure to the beta-blocker propranolol is necessary to effect gene expression. Gene expression of β2-adrenergic mRNA was decreased by the same treatment (data not shown). Addition of propranolol for 7 days for only 30 minutes daily had no effect on GIRK1 gene expression. Treatment for a shorter period of time (24 hours) also had no effect on GIRK1gene expression in our studies. The 6 day continuous exposure to propranolol caused a barely detectable decrease in GIRK2 mRNA expression and no change in GIRK4 mRNA expression levels. Longer treatment times may be necessary for gene expression changes in GIRK2 or GIRK4 similar to gene expression changes that are seen in GIRK1.
Although there were no short-term effects of beta-adrenergic agents on GIRK gene expression, we detected other cellular effects. The beta-adrenergic agonist formoterol hemifumarate stimulated potassium influx in MDA-MB-453 cells, and this influx was prevented by the GIRK channel inhibitor clozapine. NNK, a high affinity agonist for beta-adrenergic receptors  increased activation of Erk 1/2 in MDA-MB-453 breast cancer cells. Formoterol also increased activation of Erk 1/2, but to a lesser degree (data not shown). Previous studies indicated that the beta-adrenergic agonist isoproterenol stimulates growth . GIRK currents have been shown to be increased in cells stimulated with the beta-adrenergic agonist isoproterenol in rat atrial myocytes transfected with β1or β2 receptors . Heterologous facilitation of GIRK currents by β-adrenergic stimulation was also seen in rat cardiomyocytes . Two polymorphisms in the β2 and β3 adrenergic receptors were found to be correlated with a decreased risk for breast cancer , suggesting an important role of this receptor family in the genesis of breast cancer. In previous work, we demonstrated mRNA expression by RT-PCR of the β2 adrenergic receptor in the six breast cancer cell lines used in this study, but expression of β1 in all the estrogen responsive cell lines but not in two ER(-) cell lines (MDA-MB-435S and MDA-MB-453) . Further studies are needed to determine how GIRK1(+) and ER(-) breast cancers are regulated and if GIRK channel agonists and antagonists have effect on proliferation in breast cancer. It also remains to be determined if this same regulation is present in GIRK1(+) and ER(+) breast cancer malignancies. This is of particular importance since a recent report indicated that 17-β-estradiol can modulate GIRK channel activation in the brain . Future studies are also needed to determine if GIRK3 is involved in breast cancer. However, we think this unlikely because one of the functions of GIRK3 is to inhibit plasma membrane expression of other GIRK subunits .
All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines. This is the first report that implicates β-adrenergic receptors and G-protein inwardly rectifying potassium channels 1 (GIRK1) in the regulation of human breast cancer cells and suggests a potential role of the tobacco nitrosamine NNK in breast cancers expressing these regulatory pathways. Beta-adrenergic antagonists have both long term effects on gene expression and beta-adrenergic agonists have short term effect on potassium flux and cellular signaling pathways.
We gratefully acknowledge Dr. Neil Quigley (University of Tennessee Sequencing Laboratory) for his assistance with the sequencing and Kindra Walker for her assistance with cell culture, and we also acknowledge Nancy Neilsen for operation of the FACS. We also thank Alysyn Wallace-Gardner for helpful comments on the manuscript and Tommy Jordan for help with the final figures. Supported by the State of Tennessee Center of Excellence Program.
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