Abstract
Numerous studies have shown that the active form of vitamin D, 1,25(OH)(2)D(3), can exert growth inhibitory effects on human breast cancer cells and mammary tumor growth. However, the molecular mechanisms remain to be fully delineated. This study demonstrates for the first time that CCAAT enhancer-binding protein alpha (C/EBPalpha), a member of the C/EBP family of transcription factors, is induced by 1,25(OH)(2)D(3) and is a potent enhancer of VDR transcription in MCF-7 breast cancer cells. 1,25(OH)(2)D(3) was found to induce C/EBPalpha as well as VDR expression in MCF-7 cells. C/EBPalpha was not detected in MDA-MB-231 cells that are poorly responsive to 1,25(OH)(2)D(3). Antiproliferative effects of 1,25(OH)(2)D(3) and induction of VDR were observed in MDA-MB-231 cells transfected with C/EBPalpha, and knockdown of C/EBPalpha suppressed VDR and antiproliferative effects of 1,25(OH)(2)D(3) in MCF-7 cells. Transfection of C/EBPalpha in MCF-7 cells resulted in a dose-dependent enhancement of hVDR transcription. Our studies show that C/EBPalpha can bind to Brahma (Brm), an ATPase that is a component of the SWI/SNF complex, and cooperate with Brm in the regulation of hVDR transcription in MCF-7 cells. Because the levels of VDR in MCF-7 breast cancer cells correlate with the antiproliferative effects of 1,25(OH)(2)D(3) and because C/EBPalpha has been suggested as a potential tumor suppressor in breast cancer, these findings provide important mechanisms whereby 1,25(OH)(2)D(3) may act to inhibit growth of breast cancer cells. These findings also identify C/EBPalpha as a 1,25(OH)(2)D(3) target in breast cancer cells and provide evidence for C/EBPalpha as a candidate for breast cancer treatment.
Highlights
We report for the first time that C/EBP␣ is induced by 1,25(OH)2D3 in MCF-7 breast cancer cells and that C/EBP␣ cooperates with Brm, an ATPase that is a component of the SWI/SNF chromatin-remodeling complex, and is a potent enhancer of VDR transcription
C/EBP␣ Expression Is Induced by 1,25(OH)2D3 in MCF-7 Breast Cancer Cells—Shown in Fig. 1A is the 1,25(OH)2D3induced inhibition of MCF-7 cell proliferation
A major function of 1,25(OH)2D3 is to maintain calcium homeostasis, 1,25(OH)2D3 has been identified as a factor that negatively regulates the growth of a number of malignant cells in vitro and in vivo, including breast cancer cells
Summary
Materials—[␥-32P]ATP (3000 Ci (111 TBq)/mmol) was purchased from PerkinElmer Life Sciences. 5 g of the nuclear preparations from C/EBP␣transfected MCF-7 cells were incubated for 20 min at 25 °C with 2 g of poly(dI/dC) with or without unlabeled specific or nonspecific DNA competitor or C/EBP␣ antibody in binding buffer (4 mM Tris-HCl (pH 7.9), 1 mM EDTA (pH 8.0), 60 mM KCl, 12% glycerol, 12 mM HEPES, 1 mM dithiothreitol). This was further incubated with 0.5 ng of the labeled oligonucleotide probe (ϳ100,000 cpm) and incubation for 30 min at 25 °C. Statistical Analysis—Results are expressed as means Ϯ S.E., and significance was determined by analysis by Student’s t test for two-group comparison or by analysis of variance for multiple-group comparison
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