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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.

SUBMITTER: Milani C

PROVIDER: S-EPMC3637238 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Milani Cintia C Katayama Maria Lucia Hirata ML de Lyra Eduardo Carneiro EC Welsh JoEllen J Campos Laura Tojeiro LT Brentani M Mitzi MM Maciel Maria do Socorro Mdo S Roela Rosimeire Aparecida RA del Valle Paulo Roberto PR Góes João Carlos Guedes Sampaio JC Nonogaki Suely S Tamura Rodrigo Esaki RE Folgueira Maria Aparecida Azevedo Koike MA

BMC cancer 20130315

<h4>Background</h4>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.<h4>Methods</h4>Tumor samples from post-menop ...[more]

PMID: 23497279

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Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR≤0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five samples were included and were categorized according to treatment in three groups: control (A), 1,25(OH)2D3 0.5nM (B) and 1,25(OH)2D3 100nM (C).Total RNA was isolated using RNeasy kit and than was carried out according to microarray Affymetrix protocol. Data was then assessed with GeneSpring X software for background correction, normalization and summarization of raw data (CEL files) using the Robust Multi-Array Average (RMA). Five samples were included in a test and gene expression was analyzed by microarray (using SAM paired analysis). To establish a differential gene expression profile between vitamin D treated and untreated samples, SAM two class paired, provided on MEV (MultiExperiment Viewer – Boston, MA, USA) was used. Unsupervised hierarchical clustering based on Euclidean distance and average linkage was used to verify association patterns. The reliability of the clustering was assessed by the Bootstrap technique using MEV (MultiExperiment Viewer – Boston, MA, USA). Samples (16) were included in a validation group and was analyzed by qPCR.

Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR£0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five samples were included and were categorized according to treatment in three groups: control (A), 1,25(OH)2D3 0.5nM (B) and 1,25(OH)2D3 100nM (C).Total RNA was isolated using RNeasy kit and than was carried out according to microarray Affymetrix protocol. Data was then assessed with GeneSpring X software for background correction, normalization and summarization of raw data (CEL files) using the Robust Multi-Array Average (RMA). Five samples were included in a test and gene expression was analyzed by microarray (using SAM paired analysis). To establish a differential gene expression profile between vitamin D treated and untreated samples, SAM two class paired, provided on MEV (MultiExperiment Viewer – Boston, MA, USA) was used. Unsupervised hierarchical clustering based on Euclidean distance and average linkage was used to verify association patterns. The reliability of the clustering was assessed by the Bootstrap technique using MEV (MultiExperiment Viewer – Boston, MA, USA). Samples (16) were included in a validation group and was analyzed by qPCR.

Dataset Information

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

Publications

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

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