Objective
This study was undertaken to evaluate the potential role of G-protein-coupled estrogen receptor in endometrial pathology associated with tamoxifen treatment of breast cancer patients.
Study Design
We investigated whether G-protein-coupled estrogen receptor plays a role in mediating proliferating effect of tamoxifen in endometrial carcinoma cells. These results were compared with the G-protein-coupled estrogen receptor expression pattern in endometrial tissue from a cohort of 95 breast cancer patients, who received tamoxifen or another adjuvant therapy.
Results
In vitro tamoxifen significantly stimulated the mitogen-activated protein kinase phosphorylation and cell proliferation of endometrial cell lines via G-protein-coupled estrogen receptor. In vivo, there was a significant correlation between G-protein-coupled estrogen receptor expression and the tamoxifen-induced endometrial pathology ( P = .006). Moreover, G-protein-coupled estrogen receptor positivity was predictive of an earlier development of symptoms, such as bleeding or suspect endometrial thickness, induced by tamoxifen therapy ( P = .019).
Conclusion
G-protein-coupled estrogen receptor plays an important role in tamoxifen-induced endometrial abnormalities.
Estrogens are key regulators of different cellular processes involved in the physiology of female reproductive tract and mammary gland. The physiologic and pathophysiologic actions of estrogen are mediated by 2 estrogen receptors (ERs), ERα and ERβ, which belong to the steroid hormone receptor superfamily and function as hormone-inducible transcription factors. In addition to this “classic” or “genomic” effect of estrogen, there are some “rapid” or “nonclassic“ signaling effects, including stimulation of phospholipase C, cAMP, calcium, inositol phosphate, and ERK upregulation. Very recently, an orphan G-protein-coupled receptor, GPR30, was claimed to be a new membrane-bound estrogen receptor involved in the rapid nongenomic effects of estrogen. It has been demonstrated that GPR30 mediates the proliferative effects of estrogen in breast, endometrial and ovarian cancer cells (review ).
Breast cancer is the most common cancer in women and is predominantly estrogen-dependent. The endocrine treatment of choice for breast cancer premenopausal patients remains the selective estrogen-receptor modulator (SERM) tamoxifen. Although tamoxifen has an estrogen antagonistic effect in breast cancer, it acts as an estrogen agonist in the uterus. This agonistic effect of tamoxifen can stimulate cell growth and proliferation in endometrial tissue, enhancing the risk of endometrial abnormalities, including endometrial carcinoma. Interestingly, the classic ER antagonists tamoxifen and fulvestrant act as agonists for GPR30 and stimulate cell proliferation and growth. Particularly in breast and endometrial cancer cells, GPR30 activates the human epidermal growth factor receptor (EGFR) via release of heparin-bound growth factor (HB-EGF). Very recently, we have found that GPR30/EGFR crosstalk is an important component of tamoxifen agonistic activity in breast cancer cells. Moreover, it has been also shown that GPR30 mediates the tamoxifen stimulatory effects in endometrial carcinoma cells in vitro and is highly expressed in endometrial carcinoma with poor prognosis.
In this regard, the activation of GPR30 may be a possible mechanism leading to endometrial abnormalities in breast cancer patients after tamoxifen treatment. We are the first to describe the distribution of GPR30 in endometrial tissue of patients treated with tamoxifen for breast cancer and to compare it with a tamoxifen naïve cohort. The role of GPR30 for tamoxifen- induced endometrial abnormalities was confirmed in vitro, using endometrial carcinoma cell line.
Materials and Methods
Materials
All chemicals and antibiotics for a cell culture, 4-OH-tamoxifen and 17-β-estradiol (E2), were obtained from Sigma-Aldrich (Steinheim, Germany). GPR30 agonist G1 was obtained from Calbiochem (Darmstadt, Germany). GPR30 antibody was purchased from Antibodies-online (Aachen, Germany). The antibodies against total and phosphorylated MAPK and β-actin were from Cell Signaling (Frankfurt am Main, Germany). Medium was obtained from Sigma-Aldrich. GPR30 antisense oligonucleotides were purchased from MWG-Medical (Milan, Italy). Lipofectamin 2000 transfection reagent was obtained from Invitrogen (Karlsruhe, Germany).
Cell culture
Human endometrial carcinoma cells (HEC-1A) were obtained from American Type Culture Collection (Manassas, VA). They were routinely cultured in DMEM/F12 containing 5% fetal-calf serum (FCS), 100 U/mL penicillin and 100μg/mL streptomycin. To perform the experiments, the cells were seeded in 6-well plates at a density of 60,000 cells per well. To test the effect of estradiol, tamoxifen and GPR30 agonist G1, the cells were treated with phenol red-free medium supplemented with charcoal-stripped FCS 48 hours before the assay. Then, cells were incubated for 5 days with 10 nM E2, 1 nM G1, and 100 nM. At the end of the treatment, the cells were rinsed twice with saline solution and counted using a Coulter counter as described. After treatment of HEC-1A cells for 10 minutes with 100 nM tamoxifen, the phospho-MAPK (pMAPK) and total-MAPK (tMAPK) were detected by immunoblotting. Furthermore, HEC-1A cells were treated with 100 nM tamoxifen after transfection with GPR30 antisense oligonucleotides (GPR30/AS) or with control scrambled oligonucleotides (Con/AS).
Experiments with GPR30 antisense oligonucleotides
For these experiments, the cells were washed twice in phosphate-buffered saline, then transfected with 0.2 μM GPR30 antisense oligonucleotide (5′-TTGGGAAGTCACATCCAT-3′) or with 0.2 μM random control (5′-GATCTCAGCACGGCAAAT-3′). The transfection was performed with Lipofectamine 2000 transfection reagent as previously described. The experiments were performed 24 hours after transfection. The efficacy of GPR30 knocking down was detected by Western blotting.
Western blotting
Protein cell lysis and Western blotting procedures were performed as previously described. Briefly, 25 μL of the samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked with 3% dry milk in TBS/Tween and incubated for 2 hours at room temperature (RT) with antibodies against GPR30, diluted 1:1000, total or phosphorylated MAPK diluted 1: 2000, and β-actin diluted 1:10000. A peroxidase-conjugated antirabbit antibody, diluted 1:10,000, was used for a second incubation (30 minutes). West Pico Supersignal substrate (Perbio, Bonn, Germany) was left on the membrane until distinct bands had developed. A MagicMark standard (Invitrogen) was used to identify the molecular weights. The enhanced chemiluminescence membrane images were quantified using the GeneGnome and GeneTools image scanning and analysis package (Syngene, Cambridge, UK).
Patients and study design
We obtained ethical approval from Otto-von-Guericke University Ethic Committee. The study included 95 patients with primary breast cancer diagnosed between 1999 and 2004 in the Department of Obstetrics and Gynecology, University School of Medicine, Magdeburg, Germany, and in the II nd Department of Gynecology, Lublin Medical University, Lublin, Poland. They were classified into a tamoxifen group and 2 control groups. The tamoxifen group (group I) consisted of 48 patients with ER-positive breast cancer who received daily tamoxifen after breast surgery and in whom vaginal bleeding and/or suspicious endometrial thickness was observed at the periodic checkup. All these patients had undergone endometrial biopsy. A biopsy was performed if endometrial thickness of >5 mm was determined. The first control group (group II) included 22 patients who also receive daily tamoxifen, but did not have symptoms or endometrial abnormalities. The daily dose of tamoxifen for both groups was 20 mg. Group III consisted of 25 patients who did not receive adjuvant therapy with tamoxifen. The time to symptoms was calculated from the beginning of tamoxifen treatment to the first time the patients reported symptoms or suspicious endometrial thickness was observed. All patients underwent a periodic checkup with gynecologic examination, followed by ultrasound. The patients in the control groups underwent a hysterectomy because of prolapses, ovarian, pelvic, or other pathology not linked to tamoxifen therapy. Endometrial tissue developed from the curettages and hysterectomies was fixed in formalin and embedded in paraffin. The histology was classified as follows: normal/atrophic (inactive) endometrium, endometrial hyperplasia and/or endometrial polyp, and endometrial carcinoma.
Clinicopathologic data regarding diagnosis and treatment, epidemiologic factors, as well as follow-up data, were obtained retrospectively from the clinical records or the local physicians.
Immunohistochemistry
Immunohistochemistry was performed as already described. Briefly, sections of formalin-fixed and paraffin-embedded breast cancer specimens (3.0 μm thick) were mounted on SuperFrost Plus glass slides (Menzel, Braunschweig, Germany) and dried overnight. A Benchmark XT (Ventana, Unterhaching, Germany) conducted the immunostaining. The slides were incubated with affinity-purified rabbit antibody against GPR30 diluted 1:500 for 32 minutes at 37°C, after antigen retrieval with Protease I (Ventana) for 10 minutes. The reactions were visualized by 5,5′′-di-aminobenzidine (DAB) detection. The slides were counterstained with hematoxylin and coverslipped after being embedded in mounting medium.
Evaluation of GPR30 immunoreactivity
GPR30 immunoreactivity was detected in the cytoplasm and in the nucleus of the cells. Some endometrial tissues showed clear GPR30 staining pattern in the cytoplasm and weak expression in the nucleus ( Figure 1 ), whereas others demonstrated higher expression in the nucleus. In contrast, the expression of the classical ER was detected exclusively in the nucleus. No detectable cytoplasmic or plasma membrane staining was observed for ER ( Figure 1 ). The GPR30 expression was classified in accordance with the dominant immunostaining pattern. GPR30 immunoreactivity was classified as already described using the following grading system: staining extensity was categorized as 1 (<10% positive cells), 2 (10-50% positive cells), or 3 (>50% positive cells), and staining intensity was categorized as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The individual categories were multiplied to give a total immunoreactive score (IRS). IRS scores ranged between 0 and 9. GPR30 expression was classified as follows: negative (IRS between 0 and 2), low grade (IRS = 3–5), and high grade (IRS = 6-9). GPR30 protein expression was divided into 3 categories: negative, when <10% of the cells showed negative or moderate staining intensity; low grade, when 10-50% of the cells demonstrated moderate staining; high grade, when >50% showed strongly positive cells.
