Transcriptional effects of 1,25 dihydroxyvitamin D-3 physiological and supra-physiological concentrations in breast cancer organotypic culture

Abstract

Background: Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)(2)D-3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)(2)D-3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)(2)D-3 at concentrations that can be attained in vivo. Methods: Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)(2)D-3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR <= 0.1) or RT-qPCR (p <= 0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)(2)D-3 0.5nM, using RT-qPCR, western blot or immunocytochemistry. Results: 1,25(OH)(2)D-3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)(2)D-3 near physiological concentration. Genes up-modulated by both 1,25(OH)(2)D-3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)(2)D-3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)(2)D-3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)(2)D-3 0.5nM was detected. (Continued on next page) (Continued from previous page) Conclusions: In breast cancer specimens a short period of 1,25(OH)(2)D-3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)(2)D-3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.