Estrone-3-Sulfate Stimulates the Proliferation of T47D Breast Cancer Cells Stably Transfected With the Sodium-Dependent Organic Anion Transporter SOAT (SLC10A6)

Abstract

Estrogens play a pivotal role in the development and proliferation of hormone-dependent breast cancer. Apart from free estrogens, which can directly activate the estrogen receptor (ER) of tumor cells, sulfo-conjugated steroids, which maintain high plasma concentrations even after menopause, first have to be imported into tumor cells by carrier-mediated uptake and then can be cleaved by the steroid sulfatase to finally activate ERs and cell proliferation. In the present study, expression of the sodium-dependent organic anion transporter SOAT was analyzed in breast cancer and its role for hormone-dependent proliferation of T47D breast cancer cells was elucidated. The SOAT protein was localized to the ductal epithelium of the mammary gland by immunohistochemistry. SOAT showed high expression in different pathologies of the breast with a clear ductal localization, including ductal hyperplasia, intraductal papilloma, and intraductal carcinoma. In a larger breast cancer cDNA array, SOAT mRNA expression was high in almost all adenocarcinoma specimen, but expression did not correlate with either the ER, progesterone receptor, or human epidermal growth factor receptor 2 status. Furthermore, SOAT expression did not correlate with tumor stage or grade, indicating widespread SOAT expression in breast cancer. To analyze the role of SOAT for breast cancer cell proliferation, T47D cells were stably transfected with SOAT and incubated under increasing concentrations of estrone-3-sulfate (E₁S) and estradiol at physiologically relevant concentrations. Cell proliferation was significantly increased by 10^-9 M estradiol as well as by E₁S with EC₅₀ of 2.2 nM. In contrast, T47D control cells showed 10-fold lower sensitivity to E₁S stimulation with EC₅₀ of 21.7 nM. The E₁S-stimulated proliferation of SOAT-T47D cells was blocked by the SOAT inhibitor 4-sulfooxymethylpyrene.

In conclusion: The present study clearly demonstrates expression of SOAT in breast cancer tissue with ductal localization. SOAT inhibition can block the E₁S-stimulated proliferation of T47D breast cancer cells, demonstrating that SOAT is an interesting novel drug target from the group of E₁S uptake carriers for anti-proliferative breast cancer therapy.

Keywords: SLC10A6; SOAT; T47D; breast cancer; estrone-3-sulfate; proliferation; sulfate steroid; transport.

FIGURE 1

SOAT mRNA expression in breast cancer. SOAT mRNA expression was analyzed in the TissueScan^TM Breast Cancer cDNA Arrays I-IV, including 176 tumor cDNAs with different classifications (histopathology, grade, stage, and receptor status). Expression of SYMPK was used as endogenous control and ΔC_T values are depicted at the y-axis. A cut-off was set at C_T of 40. Sub-analyses were performed, including (A) tumor grade and stage, (B) receptor status for ER, PR, HER2 and triple negative breast cancer (TN), and (C) age and ethnos. As the cDNA arrays were not equally distributed for the analyzed subgroups, every single value is depicted for better clarity and additional box-whiskers-plots are given. For analysis of statistical significance, one-way ANOVA with Tukey’s multiple comparisons test was performed. Differences with p < 0.05 were not detected.

FIGURE 2

Expression of the SOAT protein in breast cancer specimen. Expression of the SOAT protein was analyzed in different breast cancer specimen by IHC with the SOAT C-13 antibody (1:100 dilution, AEC staining, hematoxylin counter stain), primary magnification × 40. Insets: Negative control without the primary antibody, primary magnification × 40. (A) SOAT expression in the ductal epithelium of normal breast tissue. (B) Strong SOAT immunoreactivity of the ductal epithelium in usual ductal hyperplasia. (C) Expression of SOAT in intraductal papilloma and expression along the ductal epithelium with strong apical lining. (D) Strong SOAT immunoreactivity in atypical ductal hyperplasia. (E) Severe immunolabeling with the SOAT antibody in intraductal carcinoma. (F) Invasive ductal carcinoma with strong and widespread SOAT expression.

FIGURE 3

Sodium-dependent uptake of E₁S into T47D-SOAT cells. (A) T47D-SOAT and T47D-control cells were incubated with 100 nM [³H]E₁S at 37°C in transport buffer containing Na⁺ or without Na⁺ (sodium-free control). Cell-associated radioactivity was analyzed at the indicated time points. (B) Transport of [³H]E₁S at 10 nM over 30 min. Data in (A,B) represent means ± SD of triplicate determinations. ^∗Significantly higher transport compared with T47D-control cells and the sodium-free controls (one-way ANOVA with Tukey’s multiple comparisons test with p < 0.05). (C) Determination of E₁S concentrations by LC-MS/MS in the cell culture medium at the end of the uptake phase. Data represent means ± SD of two independent experiments, each with triplicate determinations. ^#Significantly different between T47D-SOAT and T47D-control cells (unpaired t-test with p < 0.05).

FIGURE 4

E₁S-dependent proliferation of T47D-SOAT cells. SOAT-transfected T47D cells (T47D-SOAT, filled bars in A, black squares in B) and mock-transfected T47D cells (T47D-control, open bars in A, open squares in B) were plated at 10,000 cells/well in 24-well plates and were cultured in phenol red-free DMEM/F12 containing 5% DCC-FCS over 7 days. The culturing medium was supplemented with 10^-9 M E₂ for positive control or increasing concentrations of E₁S ranging from 10^-12 M to 10^-4 M. For negative control, cells were treated with solvent alone. Seven days after seeding, the cells were incubated with 1 μCi/ml [³H]thymidine for 2 h at 37°C, 5% CO₂, and 95% humidity. After five cycles of washing with PBS, cells were subjected to liquid scintillation counting. Data represent the means ± SD of quadruplicate determinations of three independent experiments (n = 12). (A) ^∗Significantly higher [³H]thymidine incorporation compared with negative control (one-way ANOVA with Tukey’s multiple comparisons test with p < 0.05). ^#Significantly higher [³H]thymidine incorporation in T47D-SOAT compared with T47D-control cells (p < 0.05, unpaired t-test). (B) Values were fitted to concentration response curves by non-linear regression analysis. Proliferation was determined relative to the top (100%) and bottom (0%) values that were derived from the sigmoidal dose response calculation. The EC₅₀ values were 2.2 ± 0.3 nM and 21.7 ± 2.1 nM for the T47D-SOAT and T47D-control cells, respectively. Data represent means ± SEM.

FIGURE 5

Inhibition of the E₁S-dependent proliferation of T47D-SOAT cells. Stably SOAT-transfected T47D cells (T47D-SOAT) and mock-transfected T47D cells (T47D-control) were plated at 20,000 cells/well in 24-well plates and were cultured in phenol red-free DMEM/F12 containing 5% DCC-FCS. The culturing medium was supplemented with 10^-8M E₁S, 10^-9 M E₂ or solvent alone (control). Additionally, 25 μM of the SOAT inhibitor 4-SMP were added to the culturing medium as indicated. After 6 days of cultivation, the cells were incubated with 1 μCi/ml [³H]thymidine for 2 h at 37°C, 5% CO₂, and 95% humidity. After five cycles of washing with PBS, cells were subjected to liquid scintillation counting. Data represent means ± SD of quadruplicate determinations of a representative experiment. ^∗Significantly different from solvent control (one-way ANOVA with Tukey’s multiple comparisons test with p < 0.05). ^#Significantly higher [³H]thymidine incorporation in T47D-SOAT compared with T47D-control cells (p < 0.05, unpaired t-test).

FIGURE 6

Time course of the E₁S-dependent proliferation of T47D-SOAT cells. T47D-SOAT and T47D-control cells were plated at 20,000 cells/well in 24-well plates and were cultured in phenol red-free DMEM/F12 containing 5% DCC-FCS. The culturing medium was supplemented with 10^-8 M E₁S, 25 μM 4-SMP or solvent alone. Additionally, 10^-8 M E₁S were incubated together with the SOAT inhibitor 4-SMP at 25 μM. After 2, 3, 4, 5, and 6 days of cultivation, the cells were incubated with 1 μCi/ml [³H]thymidine for 2 h at 37°C, 5% CO₂, and 95% humidity. After five cycles of washing with PBS, cells were subjected to liquid scintillation counting. Data represent the means ± SD of quadruplicate determinations of a representative experiment. ^∗Significant increase compared to day 2 (p < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). ^#Significantly higher [³H]thymidine incorporation in T47D-SOAT compared to T47D-control cells (p < 0.05, unpaired t-test).