title: 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.
  
  
  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.
authors.affiliation: Disciplina de Oncologia, LIM24, Departamento de Radiologia e Oncologia, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo 455, Sala 4124, São Paulo, SP 01246-903, Brazil.
authors.forename: Cintia
authors.initials: C
authors.lastname: Milani
authors.forename: Maria Lucia Hirata
authors.initials: ML
authors.lastname: Katayama
authors.forename: Eduardo Carneiro
authors.initials: EC
authors.lastname: de Lyra
authors.forename: JoEllen
authors.initials: J
authors.lastname: Welsh
authors.forename: Laura Tojeiro
authors.initials: LT
authors.lastname: Campos
authors.forename: M Mitzi
authors.initials: MM
authors.lastname: Brentani
authors.forename: Maria do Socorro
authors.initials: Mdo S
authors.lastname: Maciel
authors.forename: Rosimeire Aparecida
authors.initials: RA
authors.lastname: Roela
authors.forename: Paulo Roberto
authors.initials: PR
authors.lastname: del Valle
authors.forename: João Carlos Guedes Sampaio
authors.initials: JC
authors.lastname: Góes
authors.forename: Suely
authors.initials: S
authors.lastname: Nonogaki
authors.forename: Rodrigo Esaki
authors.initials: RE
authors.lastname: Tamura
authors.forename: Maria Aparecida Azevedo Koike
authors.initials: MA
authors.lastname: Folgueira
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002117:Calcitriol / Q000008:administration & dosage / Q000494:pharmacology*; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D004847:Epithelial Cells; D005260:Female; D005347:Fibroblasts; D020869:Gene Expression Profiling; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000032:analysis; D018709:Statistics, Nonparametric; D046509:Tissue Culture Techniques; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014815:Vitamins / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D014815:Vitamins; D012313:RNA; D002117:Calcitriol