Tissue-selective regulation of aromatase expression by calcitriol: implications for breast cancer therapy

Abstract

Aromatase, the enzyme that catalyzes estrogen synthesis, is critical for the progression of estrogen receptor-positive breast cancer (BCa) in postmenopausal women. We show that calcitriol, the hormonally active form of vitamin D, regulates the expression of aromatase in a tissue-selective manner. Calcitriol significantly decreased aromatase expression in human BCa cells and adipocytes and caused substantial increases in human osteosarcoma cells (a bone cell model exhibiting osteoblast phenotype in culture) and modest increases in ovarian cancer cells. Calcitriol administration to immunocompromised mice bearing human BCa xenografts decreased aromatase mRNA levels in the tumors and the surrounding mammary adipose tissue but did not alter ovarian aromatase expression. In BCa cells, calcitriol also reduced the levels of prostaglandins (PGs), major stimulators of aromatase transcription, by suppressing the expression of cyclooxygenase-2 (which catalyzes PG synthesis) and increasing that of 15-hydroxyprostaglandin dehydrogenase (which catalyzes PG degradation). The mechanism of aromatase down-regulation by calcitriol in BCa cells is therefore 2-fold: a direct repression of aromatase transcription via promoter II through the vitamin D-response elements identified in this promoter and an indirect suppression by reducing the levels of PGs. Combinations of calcitriol with three different aromatase inhibitors (AIs) caused enhanced inhibition of BCa cell growth. The combination of calcitriol and an AI may have potential benefits for BCa therapy. In addition to augmenting the ability of AIs to inhibit BCa growth, calcitriol acting as a selective aromatase modulator that increases aromatase expression in bone would reduce the estrogen deprivation in bone caused by the AIs, thus ameliorating the AI-induced side effect of osteoporosis.

Figure 1

Tissue-selective regulation of aromatase mRNA by calcitriol. A, Calcitriol decreases total aromatase mRNA in BCa cells. Total aromatase mRNA levels were determined in BCa cells treated for 24 h with 0.1% ethanol vehicle (Veh) or calcitriol (Cal, 100 nm for MDA-MB 231 and 10 nm for all other cells). *, P < 0.05 and **, P < 0.01 compared with vehicle. B, Calcitriol decreases various promoter-specific aromatase transcript levels in BCa cells. The levels of promoter-specific aromatase transcripts were determined in MCF-7 cells treated as in A. *, P < 0.05 and **, P < 0.01 compared with vehicle. C, Calcitriol increases aromatase mRNA in human osteosarcoma and ovarian cancer cells. Total aromatase mRNA levels were measured in human osteosarcoma cells (MG-63 and SaoS-2) and human ovarian cancer cells (OVCAR-3 and SKOV-3) treated as in A. **, P < 0.01 and ***, P < 0.001 compared with vehicle. D, Calcitriol increases various promoter-specific aromatase transcript levels in osteosarcoma cells. MG-63 cells were treated as in A and the levels of various promoter-specific aromatase transcripts determined. *, P < 0.05 and **, P < 0.01 compared with vehicle. E and F, Calcitriol decreases aromatase expression in 3T3-L1 preadipocytes. 3T3-L1 mouse fibroblasts representing undifferentiated preadipocytes (E, Pre-diff) were treated with ethanol vehicle (Veh) or 10 nm (Cal) for 24 h, and aromatase mRNA was measured. The cultures were then exposed to a differentiation-inducing medium with and without calcitriol addition for the final 24 h and aromatase mRNA was measured (E, Post-diff). Aromatase mRNA levels are shown as a percent of vehicle-treated control in Pre-diff cells set at 100% (E). *, P < 0.05 compared with vehicle treatment in Pre-diff cells. AP2, PPAR-γ, and perilipin mRNA levels were measured before (Pre-diff) and after (Post-diff) the induction of differentiation of 3T3-L1 cells (F). Values are shown as a percent of levels in Pre-diff cells (control) set at 100%. *, P < 0.05 compared with Pre-diff.

Figure 2

Tissue-selective regulation of aromatase activity by calcitriol. A, Calcitriol decreases aromatase activity in MCF-7 cells. MCF-7 cells were treated with vehicle (Cal, 0) or various concentrations of calcitriol (Cal, 0.1–100 nm) in serum-free medium containing the aromatase substrate T (100 nm) for 48 h. Aromatase activity was determined by measuring the concentrations of E₂ formed in the conditioned media by an EIA and expressed as picograms of E₂ formed per hour per milligram protein. **, P < 0.01 and ***, P < 0.001 compared with vehicle. B, Calcitriol decreases aromatase activity in other BCa cells. ZR-75-1 and MDA-MB 231 cells were treated with 10 or 100 nm calcitriol and aromatase activity was determined as in A. *, P < 0.05 and ***, P < 0.001 compared with vehicle. C, Calcitriol increases aromatase activity in MG-63 cells. MG-63 human osteosarcoma cells were treated with 10 and 100 nm calcitriol and aromatase activity was determined as in A. ***, P < 0.01 compared with vehicle.

Figure 3

Tissue-selective regulation of aromatase mRNA by calcitriol in mice bearing human BCa xenografts. Vehicle (Veh) or calcitriol (Cal, 0.75 μg/mouse per day for 3 d) was administered to intact (A) or OVX (B) female nude mice bearing MCF-7 xenografts. Total aromatase mRNA was measured in tumors, mammary fat tissue surrounding the tumors, and ovaries. COX-2 mRNA was measured in the tumors from intact and OVX mice (C). *, P < 0.05 compared with vehicle.

Figure 4

Calcitriol decreases PG synthesis in BCa cells. A, Calcitriol decreases COX-2 mRNA levels in BCa cells. BCa cells were treated with calcitriol as described in Fig. 1A and COX-2 mRNA levels were determined. *, P < 0.05 and ***, P < 0.001 compared with vehicle. B, Calcitriol decreases COX-2 protein levels. Western blot showing COX-2 expression in MDA-MB 231 cells treated with vehicle (Cal−) or 100 nm calcitriol (Cal+) for 48 h. COS-7 cell extracts overexpressing COX-2 and COX-1 protein served as a positive and negative control, respectively. β-Actin was used as a loading control. C, Calcitriol increases 15-PGDH mRNA levels in BCa cells. BCa cells were treated as described in Fig. 1A and 15-PGDH mRNA levels were determined. *, P < 0.05 compared with vehicle. D, Calcitriol decreases PGE₂ levels in BCa cells. MCF-7 and MDA-MB 231 cells were treated with vehicle (Veh) or calcitriol (Cal) at 10 and 100 nm, respectively, in serum-free culture media for 72 h. PGE₂ levels in conditioned media were measured. **, P < 0.01 and ***, P < 0.001 compared with vehicle.

Figure 5

Direct repression of aromatase transcription through promoter II by calcitriol. A, A schematic diagram of the aromatase promoter I.3/II. We identified two putative nVDREs (designated as proximal and distal nVDRE) in the aromatase I.3/II promoter. The putative proximal nVDRE sequence overlaps a CRE that mediates the cAMP stimulation of promoter II. B, Calcitriol suppresses basal and cAMP stimulation of aromatase I.3/II promoter activity in BCa cells. An aromatase I.3/II promoter-luciferase reporter and renilla luciferase plasmids were transiently transfected into MCF-7 cells. Cells were treated with vehicle (Veh) or 100 nm calcitriol (Cal) in the absence (control, Con) or presence 50 μm Fsk or 0.5 mm (Bu)₂cAMP for 24 h. Results are expressed as a percent of relative luciferase activity in vehicle-treated control set at 100%. **, P < 0.01 and ***, P < 0.001 compared with vehicle. ++, P < 0.01 compared with Veh + Fsk or Veh + (Bu)₂cAMP. C, Calcitriol recruits the VDR to the VDREs in the aromatase II promoter. ZR-75-1 cells were exposed to vehicle (Cal−) or 10 nm calcitriol (Cal+) for 2 h, and the cell extracts were subjected to ChIP analyses. Immunoprecipitations were carried out with IgG or a mixture of three αVDR antibodies, followed by PCR using primers designed to amplify fragments of the aromatase promoter II encompassing the proximal nVDRE and the distal nVDRE.

Figure 6

Enhanced inhibition of BCa cell growth by calcitriol-AI combinations. A, Calcitriol inhibits T stimulation of MCF-7 growth. Cells were grown in steroid-depleted media containing vehicle (control, open bar) or 100 nm T in the absence (solid bar) or presence of the indicated concentrations of calcitriol or 10 μm Cdx for 6 d. DNA contents were determined and shown as a percentage of vehicle-treated control set at 100%, which corresponded to 22.1 ± 1 μg/well. **, P < 0.01 and ***, P < 0.001 compared with control; ##, P < 0.01 and ###, P < 0.001 compared with T. B–D, Enhanced inhibition of cell growth by calcitriol-AI combinations. Cells were grown as described in A in the absence or presence of calcitriol (Cal), exemestane (Exe, 6b), anastrozole (Ana, 6c), letrozole (Let, 6d), or combinations of calcitriol with each AI at the indicated concentrations. DNA contents are shown as a percentage of vehicle-treated control set at 100%, which corresponded to 20 ± 1.2 μg/well. ***, P < 0.001 compared with control; ##, P < 0.01, ###, P < 0.001 compared with T; +, P < 0.05; ++, P < 0.01; and +++, P < 0.001 compared with the corresponding doses of calcitriol; ^, P < 0.05; ^, P < 0.01; and ^^, P < 0.01 compared with the corresponding doses of exemestane, letrozole, or anastrozole.