Calcitriol restores antiestrogen responsiveness in estrogen receptor negative breast cancer cells: a potential new therapeutic approach

Abstract

Background: Approximately 30% of breast tumors do not express the estrogen receptor (ER) α, which is necessary for endocrine therapy approaches. Studies are ongoing in order to restore ERα expression in ERα-negative breast cancer. The aim of the present study was to determine if calcitriol induces ERα expression in ER-negative breast cancer cells, thus restoring antiestrogen responses.

Methods: Cultured cells derived from ERα-negative breast tumors and an ERα-negative breast cancer cell line (SUM-229PE) were treated with calcitriol and ERα expression was assessed by real time PCR and western blots. The ERα functionality was evaluated by prolactin gene expression analysis. In addition, the effects of antiestrogens were assessed by growth assay using the XTT method. Gene expression of cyclin D1 (CCND1), and Ether-à-go-go 1 (EAG1) was also evaluated in cells treated with calcitriol alone or in combination with estradiol or ICI-182,780. Statistical analyses were determined by one-way ANOVA.

Results: Calcitriol was able to induce the expression of a functional ERα in ER-negative breast cancer cells. This effect was mediated through the vitamin D receptor (VDR), since it was abrogated by a VDR antagonist. Interestingly, the calcitriol-induced ERα restored the response to antiestrogens by inhibiting cell proliferation. In addition, calcitriol-treated cells in the presence of ICI-182,780 resulted in a significant reduction of two important cell proliferation regulators CCND1 and EAG1.

Conclusions: Calcitriol induced the expression of ERα and restored the response to antiestrogens in ERα-negative breast cancer cells. The combined treatment with calcitriol and antiestrogens could represent a new therapeutic strategy in ERα-negative breast cancer patients.

Figure 1

Immunocytochemical analysis of ERα and VDR in primary and established breast cancer cells. Representative images of cultured tumor-derived (A-C), SUM-229PE (D-F) and BT-474 (G-I) cells are shown. Tumor-derived (A) and SUM-229PE (D) cells were negative for ERα, while BT-474 was ERα positive (G). All cells were positive for VDR (B, E and H) in the cytoplasmic, nuclear and perinuclear regions (brown staining). Negative controls were carried out in the absence of primary antibody for each cell line (C, F and I). Representative pictures are displayed (20 ×).

Figure 2

Calcitriol induced ERα mRNA expression through the VDR in ERα-negative breast cancer cells. A) Cultured tumor-derived cells from five patients (1-5) with ERα-negative breast cancer and the ER-negative SUM-229PE (S) and ER-positive BT-474 (B) established cell lines were incubated with calcitriol (1X10^-7 M) or its vehicle (C, ethanol) for 24 hr. Subsequently, mRNA was extracted and real time RT-PCR (qPCR) was performed. B) Cultured breast tumor-derived cells were treated with increasing calcitriol concentrations (1X10^-10 M and 1X10^-7 M) for 24 hr. C) Cells were incubated in the absence (C) or presence of calcitriol (Cal, 1X10^-8 M), without or with a VDR antagonist (TEI, 1X10^-6 M). Results shown are the mean ± S.D. of ERα/GAPDH mRNA normalized ratio of two independent experiments per triplicate. Data were normalized to 1 for vehicle-treated cells. *P ≤ 0.05 vs. C. **P ≤ 0.05 vs. calcitriol alone.

Figure 3

Calcitriol induced ERα protein expression. SUM-229PE cells were treated with two calcitriol concentrations (Cal, 1X10^-8 M and 1X10^-7 M) and two MAPK inhibitors: U0126 (U, 10 μM) or Gefitinib (G, 0.8 μM) used as controls of ERα induction during 72 hr. Control incubations were done in the presence ethanol (C) or DMSO (D). Results were analyzed by western blots. MCF-7 (M) nuclear extracts were used as positive control for ERα and GAPDH was utilized as the loading control for normalization. Results are representative from two independent experiments.

Figure 4

Calcitriol induced a fully active ERα. Cultured breast tumor-derived cells were incubated in the absence (black bars) or presence of calcitriol 1X10^-8 M (white bars) for 48 h. Subsequently, cells were coincubated with or without calcitriol plus estradiol (E₂, 1x10^-8 M), ICI-182,780 (ICI, 1x10^-6 M) or vehicle (C) for 24 h. PRL gene expression was determined by qPCR. Results are shown as the mean ± S.D. of PRL/GAPDH mRNA normalized ratio. Data were normalized to 1 for vehicle-treated cells. *P ≤ 0.05 vs. C.

Figure 5

ERα induction restored the response to antiestrogens in ER-negative breast cancer cells. A) Cultured breast tumor-derived cells, B) SUM-229PE and C) MCF-7 were incubated in the absence (black bars) or presence of calcitriol 1X10^-8 M (white bars) for 48 h. Afterwards, cells were coincubated without (black bars) or with calcitriol (white bars) plus estradiol (E₂, 1X10^-8 M), tamoxifen (Tx, 1X10^-6 M), ICI-182,780 (ICI, 1X10^-6 M), ethanol (C), or combination of antagonists with E₂ for 6 days. Cell growth assays by the XTT colorimetric method were performed. Bars represent the mean ± S.D. Data were normalized to 100% using the activity of vehicle-treated cells. Results are representative from two independent experiments performed in sextuplicates.* P ≤ 0.05 vs. control for each group (black bars vs black control or white bars vs white control).

Figure 6

ICI-182,780 downregulated CCND1 and EAG1 gene expression in calcitriol-treated ERα-negative cells. Cultured breast tumor-derived cells were incubated in the absence (black bars) or presence of calcitriol 1X10^-8 M (white bars) for 48 h. Subsequently, cells were coincubated with or without calcitriol plus estradiol (E₂, 1x10^-8 M), ICI-182,780 (ICI, 1X10^-6 M) or its vehicle (C) for 24 hr. A)CCND1 and B)EAG1 gene expression was determined by qPCR. Results shown are the mean ± S.D. of CCND1 or EAG1/GAPDH mRNA normalized ratio. Data were normalized setting a value of 1 for vehicle-treated cells. *P ≤ 0.05 vs. C.

Figure 7

Calcipotriol induced ERα expression in ER-negative breast cancer cells. SUM-229PE cells were cultured in the presence of different calcitriol (circles) and calcipotriol (triangles) concentrations or vehicle alone (C, ethanol) for 24 hr. Afterwards, mRNA was extracted and qPCR was performed. Results are shown as the mean ± S.D. of ERα/GAPDH mRNA normalized ratio. Data were normalized setting a value of 1 for vehicle-treated cells. *P ≤ 0.05 vs. C.