Vitamin D receptor as a target for breast cancer therapy

in Endocrine-Related Cancer
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  • 1 UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
  • 2 Division of Population Health Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
  • 3 UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland
  • 4 Department of Molecular & Cellular Therapeutics, Royal College of Surgeons Ireland, Dublin, Ireland
  • 5 National Institute for Cellular Biotechnology (NICB), Dublin City University, Dublin, Ireland
  • 6 Department of Medical Oncology, St. Vincent’s University Hospital, Dublin, Ireland
  • 7 UCD Clinical Research Centre, St. Vincent’s University Hospital, Dublin, Ireland

Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.

Abstract

Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.

Introduction

The vitamin D receptor (VDR), like the oestrogen receptor (ER), progesterone receptor (PR) and the androgen receptor (AR), is a member of the nuclear family of steroid hormone transcriptional regulators (Leyssens et al. 2013, Narvaez et al. 2014, Feldman et al. 2015). VDR is activated after the binding of the active form of vitamin D known as 1α,25(OH)2-vitamin D3 or calcitriol. Ligand binding results in heterodimerisation with the retinoid X receptor (RXR), which is followed by binding to vitamin D response elements on DNA. After the recruitment of multiple transcriptional regulating proteins such as nuclear receptor co-activators, the calcitriol-bound VDR/RXR complex begins to alter gene expression (Narvaez et al. 2014). VDR has been shown to be both an activator and repressor of transcriptional activity, i.e., its target genes may be upregulated or downregulated (Haussler et al. 2013, Campbell 2014, Christakos et al. 2016).

Unlike ER and AR that have been widely exploited as therapeutic targets in cancer (i.e., ER in breast cancer (EBCTCG et al. 2011) and AR in prostate cancer (Valenca et al. 2015)), less work has been carried out on targeting VDR. There are, however, several preclinical studies suggesting that targeting VDR with calcitriol or its synthetic analogues may have anti-cancer potential (Narvaez et al. 2001, Flanagan et al. 2003, Kasiappan et al. 2014, Lungchukiet et al. 2014, Chen et al. 2015, Murray et al. 2015). In this investigation, using a large panel of breast cancer cell lines that included ER-positive, HER2-positive and triple-negative (TN) (i.e., negative for ER, PR and not HER2 amplified) cells, we compared the anti-proliferative effects of the low calcaemic-inducing vitamin D analogue, inecalcitol, with that of calcitriol.

Inecalcitol (also known as TX-522 or 19-nor-14-epi-23-yne-1α,25-(OH)2-vitamin D3) (Hybrigenics, Paris, France) is a 14-epi-analogue of calcitriol (1α,25(OH)2-vitamin D3) that was previously shown to be at least 10-fold more potent than calcitriol in decreasing growth or inducing apoptosis in a number of different types of cancer cells (Verlinden et al. 2000, Okamoto et al. 2012, Ma et al. 2013). Although more potent than calcitriol in blocking cancer cell growth, it was less calcaemic in the animal models investigated (Verlinden et al. 2000, Swami et al. 2012). This enhanced potency in blocking cell proliferation when combined with its lower calcaemic-inducing properties prompted us to further investigate inecalcitol as a potential new therapeutic for breast cancer.

Here, we show that the anti-proliferative effects of both calcitriol and inecalcitol are dependent on the molecular subtype of the breast cancer cell line as well as on the endogenous concentration of VDR. We also found that the IC50 values of inecalcitol for growth inhibition were consistently lower than those for calcitriol across the cell line panel investigated. In addition, using RNA-seq analysis, we found substantial but not complete overlap between the genes differentially regulated by calcitriol and inecalcitol.

Materials and methods

Cell lines and reagents

A panel of 15 breast cancer cell lines representing all the main molecular subtypes of breast cancer were investigated. These included: Hs578t(i8), MDA-MD-468, MDA-MD-231, HCC1937, HCC1143, BT20, BT549 (all TN); MDA-MD-453, SKBR3, BT474, JIMT-1 (all HER2-positive) and T47D, Cama1, ZR-75-1 and MCF-7 (all ER-positive or luminal). Apart from Hs578t(i8) cells that were supplied by Dr Susan McDonnell, University College, Dublin (Hughes et al. 2008), all other cell lines were purchased from the American Type Culture Collection (ATCC).

Cell lines were maintained through continued passaging at 37°C with a humidified atmosphere of 5% CO2. All media was supplemented with 10% foetal bovine serum (FBS) (Invitrogen Life Technologies), 1% penicillin/streptomycin (Invitrogen Life Technologies) and 1% Fungizone (Invitrogen Life Technologies). BT549 cells were maintained in RPMI-1640 supplemented with 0.023 IU Insulin (Sigma-Aldrich), 10 mM Hepes (Sigma-Aldrich), 1.5 g/L sodium bicarbonate (Sigma-Aldrich) and 1 mM sodium pyruvate (Sigma-Aldrich). ZR-75-1 cells were maintained in DMEM supplemented with 1 nM oestradiol (Sigma-Aldrich). Cell line identity was confirmed by analysis of short-term repeat loci, and cells were routinely tested for mycoplasma infection.

Cell viability assays

Cell proliferation was assessed using 3-(4,5-dimethyl­thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay as previously described (McGowan et al. 2013). To test the effect of calcitriol (Selleckchem, Munich, Germany) or inecalcitol (Hybrigenics, Paris, France) on proliferation, cells were plated at a density of 1 × 103/well in 96-well flat-bottomed plates (Sigma-Aldrich). After overnight incubation, quadruplicate wells were treated with concentrations of compounds ranging from zero to 20 μM. After 5 days, 0.5 mg/mL MTT was added to each well and incubated at 37°C for 5 h. Media was aspirated, and 200 μL of DMSO was added to each well, for 5 min. Absorbance was measured at a wavelength of 550 nm on a microplate reader (Multiscan Ascent, Labsystems).

Western blotting

Protein lysates (50 μg) were separated on a 10% polyacrylamide gel (Bioscience) (200 volts for 60 min). After electrophoresis, protein was transferred to a nitrocellulose membrane (Millipore) and blocked for 2 h at room temperature in 2% low-fat milk (Marvel instant dried skimmed milk) in Tris-buffered saline/0.1% Tween 20 (TBST). The membranes were then probed overnight at 4°C with a mouse monoclonal anti-VDR antibody (clone D-6, Santa Cruz). After 3 washes for 10 min in TBST, membranes were incubated with a horseradish peroxidise-conjugated secondary anti-mouse antibody (Cell Signalling Technology) or anti-rabbit antibody (Cell Signalling Technology) for 2 h at room temperature. Signals were developed using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific) and exposure to X-ray film (Fujifilm). As a control measure for equal loading, blots were re-probed for GADPH. Positivity was defined as a visible band at the appropriate molecular mass location on the blot.

Enzyme-linked immunosorbent assay

Quantification of VDR was carried out using the Human VDR ELISA kit (Cloud Clone Corp., Houston, TX, USA; SEA475Hu), as per manufacturer’s protocol. Absorbance was measured at a wavelength of 450 nm on a microplate reader (Multiscan Ascent, Labsystems).

Measurement of cell migration

Transwell migration assay was performed by seeding cells at a density of 2.5 × 105/500 µL in the upper compartment of a cell permeable membrane insert (8 μm pore size; BioCoat, BD Biosciences). The lower chamber was filled with fibroblast-conditioned medium (supplied by Prof. Ursula Fearon, University College Dublin). After a 5 h incubation period, the cells were treated with 2 µM calcitriol or 4 nM inecalcitol. After 5 days of incubation, non-migrated cells in the upper chamber were removed with a PBS-soaked cotton swab. This was followed by fixation using 1% glutaraldehyde (Sigma-Aldrich) and staining with 0.1% crystal violet (Pro-Lab Diagnostics, Birkenhead, UK). Cells fixed on the lower face of the Matrigel chambers were counted under a light microscope at a magnification of ×40.

RNA-seq analysis

MCF-7 cells were treated with 2 µM calcitriol or 4 nM inecalcitol in triplicate for 6 h. DMSO and ethanol acted as controls for calcitriol- and inecalcitol-treated cells, respectively. RNA was isolated using RNeasy Mini kits (Qiagen) according to the manufacturer’s instructions. Quantity and purity were determined using a Bioanalyzer (Agilent). Libraries were prepared using the Illumina TruSeq stranded mRNA library prep kit according to the manufacturer’s instructions (Illumina). Sequencing was performed using an Illumina HiSeq 2500 to produce 51 bp single-end reads, and quality control was conducted using FASTQC. (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to human genome, version 19, using the sequence aligner subread (Liao et al. 2013) via the bioconductor R package Rsubread (https://bioconductor.org/packages/). The data were scale normalised using the TMM (trimmed mean of M values (TMM)) normalisation method (Robinson & Oshlack 2010). Differential expression was determined using the ebayes function of the R package LIMMA (Smyth 2004).

An adjusted P value <0.05 and a fold-change greater than 1.4-fold was considered significant. The P values were adjusted using the Benjamini and Hochberg method (Benjamini & Hochberg 1995). The package LIMMA was chosen here for differential expression analysis as it has been shown to be particularly robust when dealing with small sample sizes (Seyednasrollah et al. 2015). Gene ontology (GO) analysis was performed using the R package goseq (Young et al. 2010), a package specifically designed for performing GO analysis on RNA-seq data, so as to overcome the inherent bias in RNA-seq data for over-detecting long and highly expressed transcripts. For comparison across public datasets, gene lists as published by the original authors were used. All calculations were carried out in the R statistical environment (https://cran.r-project.org/).

qPCR confirmation of SNAI2 and MYBPH expression

Total RNA isolated using the RNeasy Mini kit (Qiagen) was transcribed into cDNA (0.5 µg of template) using the RT2 First Strand kit (Qiagen). RT-PCR analysis of SNAI2 and MYBPH mRNA was performed in triplicate using the RT2 qPCR Primer Assay (Qiagen) and GAPDH as housekeeping gene. Cycle threshold (Ct) values were normalised using the housekeeping gene, and the fold-change gene expression was calculated using the ∆∆Ct method.

Evaluation of the effect of VDR expression on prognosis in patients with different molecular subforms of breast cancer

To establish a potential prognostic value for VDR in breast cancer patients, we evaluated a pooled database of 12 publicly available breast cancer datasets (n = 2019 patients) containing gene expression data (Madden et al. 2013), as previously described (Caiazza et al. 2016). The median expression level of VDR was used to dichotomise the data, and disease-free survival (DFS) was chosen as the survival endpoint.

Statistical analysis

IC50 data were analysed using Prism, version 5.0b software (GraphPad Software). The correlation coefficients were calculated using the Spearman’s non-parametric test. The IC50 values of calcitriol and inecalcitol were compared using Students t-test. The IC50 concentration for each compound was determined using CalcuSyn software (Biosoft). All experiments were carried out in triplicate, and the results are presented as means ± s.e.m. (standard error of the mean). IC50 values >20 µM were extrapolated values. The effects of calcitriol and inecalcitol on migration of the cell lines were also analysed using Student’s t-test. P values <0.05 were considered significant.

Results

Comparative effects of calcitriol and inecalcitol on cell growth inhibition in a panel of breast cancer cell lines

Table 1 compares the IC50 values for calcitriol and inecalcitol in a panel of 15 breast cancer cell lines. Across the panel, IC50 concentrations for calcitriol ranged from 0.1 µM to >20 µM, whereas the corresponding values for inecalcitol varied from 2.5 nM to 63.5 nM. Overall, IC50 values for inecalcitol were significantly lower for inecalcitol than those for calcitriol (P < 0.001) (Fig. 1A), i.e., inecalcitol was significantly more potent in inhibiting proliferation than calcitriol. Irrespective of the cell line, the increased potency for inecalcitol vs calcitriol was >18-fold.

Figure 1
Figure 1

Effect of calcitriol or inecalcitol on cell line growth and relationship between response to calcitriol or inecalcitol treatment and VDR protein levels. (A) Comparative IC50 values for breast cancer cell lines treated with calcitriol or inecalcitol. (B) and (C) scatter plot representing the relationship between IC50 values for calcitriol (B) or inecalcitol (C) and VDR status. Data were analysed using Student t test.

Citation: Endocrine-Related Cancer 24, 4; 10.1530/ERC-16-0463

Table 1

Comparative IC50 concentration for calcitriol and inecalcitol, using MTT assay.

Cell lineSubtypeMean IC50 (µM) ± s.e.m.Mean IC50 (nM) ± s.e.m.FD
CalcitriolInecalcitol
JIMT-1HER2+0.17 ± 0.019.37 ± 1.1218.2
T47DLuminal0.78 ± 0.088.20 ± 1.0095.1
MCF-7Luminal1.83 ± 0.694.2 ± 1.73435.7
Cama1Luminal2.78 ± 1.656.53 ± 0.71425.7
HCC 1937TN6.75 ± 1.99.80 ± 0.61688.7
MDA-MB-453HER2+8.82 ± 2.1422.95 ± 4.28384.3
MDA-MB-468TN9.93 ± 2.8245.35 ± 4.28218.9
ZR751Luminal>202.54 ± 0.66>1000
BT20TN>2032.9 ± 8.16607
BT474HER2+>2010.60 ± 0.17>1000
HCC 1143TN>2023.89 ± 3.25837
Hs578t(i8)TN>2044.05 ± 1.59454
MDA-MB-231TN>2063.51 ± 9.77314
SKBR3HER2+>2052.22 ± 17.01382
BT549TN>2036.31 ± 7.00550

All assays were performed in triplicate.

FD, fold difference; TN, triple-negative.

Relationship between response to calcitriol or inecalcitol and cellular VDR level

As calcitriol is believed to mediate its actions via its specific receptor, we related IC50 values for calcitriol and inecalcitol to the cell line concentration of VDR. To do this, VDR levels were determined by both Western blotting and ELISA (Supplementary Fig. 1A and B, see section on supplementary data given at the end of this article). Overall, a significant correlation was found between VDR levels determined using the 2 methods (P = 0.0009, r = 0.7634, n = 15) (Supplementary Fig. 1C). The BT-549 cell line, however, lacked a visible VDR band after Western blotting but was VDR positive using ELISA. This discrepancy may relate to the specificity of the antibodies used in the different assays. The antibody used in the Western blotting binds to amino acids 344–424, i.e., towards the C-terminal end of the VDR. This region of VDR may be missing in BT549 cells and if so, no protein would be detected using Western blotting. We are unable to establish the identity of the epitopes recognised by the antibodies in the ELISA, as this information is proprietary. However, if these antibodies bind to a different region in VDR than the antibody used for Western blotting, this might explain our positive finding with ELISA but undetectable levels using Western blotting.

Using Western blotting, VDR was found to be expressed in 10 of the 15 cells lines investigated and absent in 5 cell lines (Supplementary Fig. 1A). As shown in Fig. 1B, IC50 values for calcitriol were significantly lower in cell lines expressing VDR than those in cell lines negative for VDR (P = 0.0007, n = 15) (Fig. 1B). Indeed, all the VDR-negative cell lines were resistant to calcitriol with IC50 values >20 µM (Table 1). Similarly, response to inecalcitol was significantly greater in VDR-positive vs VDR-negative cell lines (P = 0.008) (Fig. 1C). However, in contrast to the situation with calcitriol, inecalcitol appeared to be growth inhibitory in VDR-negative cell lines.

Effect of molecular subtype of cell line on response to calcitriol and inecalcitol

Response to both calcitriol and inecalcitol was related to the ER, AR and HER2 status of the cell lines. As shown in Fig. 2, the IC50 values for both compounds were significantly lower in ER-positive cell lines than those in ER-negative cell lines, (for calcitriol, P = 0.0430; for inecalcitol, P = 0.005) (Fig. 2A and B). In contrast, response to calcitriol and inecalcitol was independent of both the AR (Fig. 2C and D) and HER2 status (Fig. 2E and F). In an attempt to explain why ER-positive cell lines were more sensitive than ER-negative cell lines to both calcitriol and inecalcitol, we compared VDR levels determined by ELISA in the 2 groups of cell lines. Although there was a trend for higher concentrations of VDR in ER-positive compared to ER-negative cell lines, this difference did not reach statistical significance (P = 0.133). The failure to reach statistical significance may be due to the relatively small number of cell lines investigated.

Figure 2
Figure 2

Relationship between the response to calcitriol or inecalcitol treatment and the molecular subtype of breast cancer cell line. (A, B, C, D, E and F) Scatter plots representing the relationship between response to treatment with calcitriol (A, C and E) or inecalcitol (B, D and F) and the molecular subtype of breast cancer. Scatter plots representing ER+ and ER− cell lines (A and B), AR+ and AR− cell lines (C and D) or HER2+ or HER2− cell lines (E and F). Data were analysed using the Student’s t test. Data points represent the mean of three independent experiments.

Citation: Endocrine-Related Cancer 24, 4; 10.1530/ERC-16-0463

Effect of calcitriol and inecalcitol on migration

In addition to enhanced proliferation, cell migration is essential for cancer progression, especially for invasion and metastasis. We therefore investigated the effects of treatment with calcitriol (2 µM) or inecalcitol (4 nM) on migration using the Transwell migration assay (Fig. 3A, B, C and D). With MCF-7 cells, calcitriol reduced migration by 41.9% (P = 0.015) vs 56.3% (P = 0.012) after treatment with inecalcitol; with BT20 cells, the corresponding reductions in migration were 45.9% (P = 0.045) and 57.6% (P = 0.007), whereas with Hs578t(i8), the reductions in migration were 40.3% (P = 0.043) and 49.3% (P = 0.001), respectively. Unlike our findings with MCF-7, BT20 and Hs578t(i8) cells, calcitriol failed to significantly reduce migration in MDA-MB-453 (P = 0.079). In contrast to calcitriol, treatment with inecalcitol significantly reduced migration in MDA-MB-453 cells (reduction in migration, 52.9%, P = 0.033). Overall, across the 4 cell lines, inecalcitol had a significantly greater effect on migration than calcitriol (P = 0.0018).

Figure 3
Figure 3

Effect of calcitriol or inecalcitol on cell migration. Bar chart representing the percentage cell migration as determined by the Transwell migration, as well as a representative image of Transwell insert (×40) for MCF-7 (A), BT20 (B), Hs578t(i8) (C) or MDA MB 453 cell lines. Cells were treated with either 2 µM calcitriol or 4 nM inecalcitol. Percent migration was determined by quantifying the number of cells on the underside of the insert and normalising to vehicle control. All experiments were carried out in triplicate. Data were analysed using Paired t test. A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0463.

Citation: Endocrine-Related Cancer 24, 4; 10.1530/ERC-16-0463

Effect of calcitriol and inecalcitol on gene expression

In an attempt to establish if calcitriol and inecalcitol had a similar impact on gene expression, we compared the effects of the 2 compounds on MCF-7 cells using RNA-seq. Overall, 106 genes were found to be significantly regulated after treatment with 2 µM calcitriol and 98 after the addition of 4 nM inecalcitol (Supplementary Tables 1 and 2 for full list of altered genes and Table 2 for the top 20 differentially regulated genes). The majority of the genes were upregulated, which is consistent with previous studies (Goeman et al. 2014, Sheng et al. 2015, Simmons et al. 2015). Of the total number of genes significantly differentially regulated by either compound, 77 were altered by both. In addition, there was a strong correlation in the fold changes of these 77 genes between the two treatments (r = 0.9331, P = 0.0001) (Supplementary Fig. 2). Indeed, the 2 genes showing the greatest extent of upregulation were the same for both compounds, i.e., cytochrome p450, family 24, subfamily a, polypeptide 1 (CYP24A1) and src homology 2 domain-containing E (SHE). Thirty-two genes, however, were significantly differentially regulated by calcitriol that were not altered by inecalcitol, whereas 24 genes were significantly differentially regulated by inecalcitol but not by calcitriol (Supplementary Table 1).

Table 2

Top 20 upregulated genes in MCF-7 cells following treatment with calcitriol or inecalcitol.

RankCalcitriol treatedInecalcitol treated
Gene IDGene symbolFold changeAdjusted P-valueGene IDGene symbolFold changeAdjusted P-value
11591CYP24A151.402.51E-271591CYP24A1160.031.94E-26
2126669SHE15.023.99E-05126669SHE19.141.22E-04
31417CRYBB37.561.14E-0254436SH3TC19.902.22E-08
455503TRPV66.592.68E-023497IGHE8.764.19E-07
5152015ROPN1B6.584.78E-0227128CYTH46.631.12E-02
63497IGHE6.271.62E-0755503TRPV66.402.62E-02
7139735ZFP926.193.57E-02553137LOC5531375.412.45E-02
8643904RNF2225.891.52E-024608MYBPH4.806.60E-03
95593PRKG25.361.38E-0627201GPR784.712.06E-02
1054436SH3TC14.702.38E-075593PRKG24.167.91E-05
11154865IQUB4.643.86E-02219855SLC37A23.722.60E-04
1284559GOLGA2P2Y4.554.78E-027113TMPRSS23.704.64E-18
13646643SBK24.271.40E-0410461MERTK3.501.25E-07
144880NPPC4.053.86E-025653KLK63.451.86E-11
15219855SLC37A23.251.76E-04158326FREM13.441.41E-03
1680031SEMA6D3.231.33E-0280031SEMA6D3.412.08E-02
177113TMPRSS23.201.81E-178111GPR683.317.81E-08
1810461MERTK3.203.18E-07124872B4GALNT23.281.83E-02
195653KLK63.172.09E-113708ITPR13.169.87E-12
20389602LOC3896023.121.20E-029459ARHGEF63.026.95E-06

Genes unique to treatment with either calcitriol or inecalcitol are indicated in bold.

We specifically investigated if either calcitriol- or inecalcitol-modulated expression of VDR. Using RNA-seq, neither compound was found to alter mRNA levels of VDR in MCF-7 cells. Similarly, using RT-PCR, VDR expression was not found to be modulated by either calcitriol or inecalcitol in this cell line.

Gene ontology (GO) terms significantly enriched in differentially regulated genes

Supplementary Table 3 lists the most significantly enriched GO terms after treatment with either calcitriol or inecalcitol. Of these, 10 were found be significantly enriched after treatment with both compounds. These included pathways involved in muscle cell migration, protein citrullination, negative regulation of cellular processes, negative regulation of locomotion, neuron development and citrulline biosynthetic processes. Some GO terms included in the top 20 enriched list, however, were significantly modulated by calcitriol but not by inecalcitol, i.e., bone morphogenesis, ossification and eyelid development. On the other hand, the GO terms, myeloid development, cell development, cell surface receptor signalling pathways and cell communication were amongst the top 20 significantly enriched terms after treatment with inecalcitol but not after treatment with calcitriol.

Comparison of differentially regulated genes across public transcriptomic studies

Table 3 compares the differentially regulated genes observed in this study with those of previously reported transcriptomic studies involving calcitriol (Goeman et al. 2014, Sheng et al. 2015, Simmons et al. 2015). It is important to state that these different studies for the most part used different cell lines, different concentrations of calcitriol different treatment times and different techniques for investigating gene expression (Supplementary Table 4). Despite these differences, 23, 14 and 27 of the genes whose expression was significantly altered after calcitriol treatment in the present investigation were also significantly modulated in the study of Goeman and coworkers (Goeman et al. 2014), Sheng and coworkers (Sheng et al. 2015) and Simmons and coworkers (Simmons et al. 2015), respectively. Only 4 genes however, were found to be differentially regulated across all studies after treatment with calcitriol, i.e., CYP24A1, transmembrane protease serine 2 (TMPRSS2), calponin-like transmembrane (CLMN) and EFL1 elongation factor-like GTPase 1 (EFTUDE). Interestingly, the same 4 genes were also found to be altered with inecalcitol in this study. Of note, CYP24A1 was the top regulated gene in all studies.

Table 3

Comparison of genes differentially regulated by calcitriol or inecalcitol in the present study vs those available in public datasets.

Gene symbolGene IDCalcitriolInecalcitolGoeman et al. (2014)Sheng et al. (2015)Simmons et al. (2015)
ARHGEF694592.153.021.54NS2.35
C2orf54799191.721.711.65NSNS
CLCF1235291.661.652.641.60NS
CLMN797892.642.792.042.373.30
CYP24A1159151.40160.03533.74243.00277.37
EFTUD1796312.092.031.722.492.22
FOS23532.222.554.35NSNS
IGFBP334861.591.721.92NS2.41
N4BP3231381.551.581.641.80NS
PGM2L12832091.951.831.87NS2.50
PISD237611.551.741.62NS2.10
TMEM371407382.091.833.182.15NS
TMPRSS271133.203.702.813.402.23
TRIM56818441.581.621.77NSNS
TRPV6555036.596.402.91NS6.74
TSKU259872.031.922.45NS2.01
ZNF16577181.541.451.54NSNS
G6PD25391.541.63NS1.991.61
GFPT29945−1.56−1.44NS−1.99NS
ITGA236731.611.60NS2.102.46
ITPR137082.743.16NS1.842.77
KLK656533.173.45NS7.2710.42
LINGO1848941.781.91NS1.68NS
SHE12666915.0219.14NS2.36NS
ACOX383101.812.01NSNS1.54
DHRS392492.052.03NSNS1.58
ENPEP20281.711.70NSNS1.55
FSTL4231051.501.63NSNS2.71
MERTK104613.203.50NSNS3.82
PADI3517022.082.38NSNS1.75
PRKG255935.364.16NSNS3.07
SMOX544981.731.73NSNS1.55
CEBPB10511.57NS0.98NS1.52
DOCK111398181.77NSNSNS1.74
PACSIN2112521.43NSNSNS1.61
RNF144B255488−1.46NSNSNS−1.67
RNF144B255488−1.46NSNSNS−1.77
DUSP1011221NS1.48NSNS1.51
EGLN3112399NS−1.43NSNS−1.65
EGLN3112399NS−1.43NSNS−1.75
AHRR574911.69NS1.23NSNS
CLSTN397461.47NS0.64NSNS
OSR21160391.45NS1.19NSNS
RNF2226439045.89NS2.20NSNS
SLC16A591212.05NS1.85NSNS

Values presented are fold-changes. Values in bold represent genes that were altered across all studies.

NS, not significant.

Confirmation of the effect of calcitriol and inecalcitol on gene expression using candidate genes of interest

Although the RNA-seq analysis showed major overlap in the genes regulated by calcitriol and inecalcitol in MCF-7 cells, a small number were specifically regulated by only one of the compounds. Thus, snail family zinc finger 2 (SNAI2) was found to be upregulated by calcitriol only (Supplementary Table 1). On the other hand, expression of myosin-binding protein H (MYBPH) was increased by inecalcitol but not by calcitriol (Supplementary Table 1). We decided to confirm and extend these findings using qPCR. As shown in Fig. 4A, using RT-PCR, the addition of calcitriol increased the expression of SNAI2 not only in MCF-7 cells but also in 3 other cell lines, i.e., in CAMA1 and T47D an MDA-MB-453 cells. Calcitriol, however, did not increase SNAI2 expression in BT20 or Hs578t(i8) cells. Similarly, no increase in SNAI2 was seen in any of the 6 cell lines investigated after the addition of inecalcitol. Indeed increasing the concentration of inecalcitol 4-fold (i.e., up to 16 nM) failed to significantly upregulate SNAI2 expression (data not shown).

Figure 4
Figure 4

Effect of calcitriol or inecalcitol treatment on the expression of SNAI2 and MYBPH. (A) and (B) bar chart representing the fold-change in expression of SNAI2 (A) or MYBPH (B) after either 2 µM calcitriol (grey) or 4 nM inecalcitol (white) treatment of a panel of breast cancer cell lines. Data were analysed using the Student’s t test. All experiments were carried out in triplicate.

Citation: Endocrine-Related Cancer 24, 4; 10.1530/ERC-16-0463

In contrast to SNAI2, using RNA-seq, MYBPH was found to be upregulated in MCF-7 cells by inecalcitol but not by calcitriol. Consistent with the RNA-seq data, RT-PCR analysis confirmed that MYBPH was upregulated by inecalcitol in MCF-7 cells (Fig. 4B). Similarly, RT-PCR showed that expression of MYBPH was increased in 5 other cell lines after treatment with inecalcitol. In contrast, calcitriol had no effects in 5 of these cell lines. Although, using RT-PCR, calcitriol was found to upregulate MYBPH expression in MCF-7 cells, the magnitude of this effect was small (Fig. 4B).

Relationship between VDR expression and prognosis in breast cancer

To extend our preclinical findings, we related tumour VDR levels to outcome in a public database of breast cancer survival and gene expression data (Madden et al. 2013). Using the median expression to dichotomise the gene expression data and DFS as the survival endpoint, VDR expression was not significantly associated with survival in breast cancer as a whole (Fig. 5A). Similarly, VDR was not significantly associated with DFS in the HER2-positive, basal or luminal B molecular subtypes (Fig. 5B, C and D). In contrast, high levels of VDR predicted good outcome in the luminal A patients (P = 0.0056, HR = 0.74, n = 1035) (Fig. 5E) and in the luminal A patients known to be treated with adjuvant tamoxifen (P = 0.0003, HR = 0.27, n = 273) (Fig. 5F).

Figure 5
Figure 5

Effect of VDR expression on disease-free survival in breast cancer. The prognostic value of VDR expression in breast cancer was determined using the BreastMark database (Madden et al. 2013). Kaplan–Meier survival curves for (A) total population (n = 2592, HR = 0.9318, P = 0.290), (B) HER2 subgroup (n = 325, HR = 0.9515, P = 0.7640), (C) basal subgroup (n = 394, HR = 0.8932, P = 0.4347), (D) luminal B subgroup (n = 617, HR = 1.026, P = 0.8149), (E) luminal A subgroup (n = 1035, HR = 0.7393, P = 0.0056) or (F) luminal A treated with adjuvant tamoxifen (n = 273, HR = 0.2726, P = 0.0003). A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0463.

Citation: Endocrine-Related Cancer 24, 4; 10.1530/ERC-16-0463

Discussion

In this investigation, we showed that both calcitriol and the low calcemic-inducing analogue, inecalcitol inhibited breast cancer cell growth in a cell line-dependent manner. Although previous studies also showed that calcitriol as well as some of its specific analogues blocked the growth of breast cancer cells, almost all of these reports used only one or a small number of cell lines (Vink-van Wijngaarden et al. 1994, Welsh 1994, Simboli-Campbell et al. 1996, Swami et al. 2000, 2012, Verlinden et al. 2000, Narvaez et al. 2001, Flanagan et al. 2003, Lundqvist et al. 2014). Furthermore, most of these earlier studies failed to measure the IC50 values for growth inhibition by calcitriol or its analogues.

In contrast to the limited number of cell lines used in most of the previous preclinical studies evaluating the cell growth inhibitory potential of calcitriol, our investigation used 15 cell lines representing all the different molecular subtypes of breast cancer, i.e., ER positive, HER2 positive and TN. Furthermore, unlike most previous studies, we measured IC50 values for all the cell lines investigated, and we related response to calcitriol and inecalcitol to the endogenous concentration of the VDR. Although not unexpected, we found that IC50 values for both compounds were significantly lower in VDR-positive cells vs VDR-negative cells.

However, the growth of some cell lines that had undetectable levels of VDR by both Western blotting and ELISA was inhibited by inecalcitol. A possible explanation for this finding is that inecalcitol may mediate some of its effects independent of VDR. Across the full panel of 15 cell lines, however, IC50 values for inecalcitol were lower in the VDR-positive cell lines compared to those in the VDR-negative cell lines, suggesting that the predominant anti-proliferative effects of inecalcitol occurs via VDR.

The finding that VDR-positive cell lines were more sensitive than VDR-negative cell lines to both calcitriol and inecalcitol suggests that VDR levels may be a predictive biomarker for response to these compounds. Although this result might be expected, we are not aware of a previous study that related VDR levels with sensitivity to calcitriol or any of its analogues in a relatively large panel of cancer cell lines.

The extent of growth inhibition, however, depended not only on the endogenous VDR level but also on the molecular subtype of the cell line, i.e., ER-positive cell lines were more sensitive to both compounds than ER-negative cell lines. This enhanced response in ER-positive cell lines, may relate, at least in part, to the trend towards higher levels of VDR in these cell lines compared to ER-negative cells. In contrast to the enhanced response in ER-positive vs ER-negative cell lines, response to both compounds was independent of HER2 and AR status.

Although multiple studies have previously reported that calcitriol has cancer cell growth inhibitory properties (Narvaez et al. 2001, Flanagan et al. 2003, Swami et al. 2012, Kasiappan et al. 2014, Lungchukiet et al. 2014, Chen et al. 2015, Murray et al. 2015) and thus theoretically might be used to treat cancer, it is unlikely to be used clinically due to its hypercalcaemia-inducing activity at the concentrations necessary to inhibit cancer cell growth. In an attempt to circumvent the induction of hypercalcaemia, several low calcemic-inducing vitamin D analogues have been synthesised (for review, see Leyssens et al. 2013). Of the synthetic low calcemic vitamin D analogues, inecalcitol is the most clinically advanced for cancer treatment. This compound was previously reported to inhibit the growth of MCF-7 cells both in vitro and in an animal model (Verlinden et al. 2000, Swami et al. 2012). As mentioned in the Introduction section, previous studies reported that inecalcitol was at least 10-fold more potent than calcitriol in blocking cancer cell growth and furthermore was less calcemic in the animal models investigated (Verlinden et al. 2000, Swami et al. 2012).

Here, we not only confirm this greater potency of inecalcitol vs calcitriol in MCF-7 cells but extend the finding across a panel of 15 breast cancer cell lines. Indeed, our IC50 values for inecalcitol were at least 18-fold lower than those found with calcitriol. Inecalcitol has already undergone a phase I clinical trial in combination with docetaxel in patients with advanced prostate cancer (Medioni et al. 2014). Of the 54 patients treated in this early phase trial, 50 were assessable for response based on PSA measurement. In these assessable patients, PSA levels decreased by ≥30% within 3 months of initiation of treatment in 85% of cases. Overall, the treatment was well tolerated except when the highest concentration of inecalcitol was administered (i.e., 4000 μg bid). Furthermore, inecalcitol has recently received orphan drug designation status for the treatment of acute myeloid leukaemia and chronic lymphocytic leukaemia in both Europe and the USA (http://www.ema.europa.eu/docs/en_GB/document_library/Orphan_designation/2014/03/WC500163572.pdf, https://www.accessdata.fda.gov/scripts/opdlisting/oopd/detailedIndex.cfm?cfgridkey=432914, http://www.ema.europa.eu/docs/en_GB/document_library/Orphan_designation/2015/08/WC500192011.pdf, https://www.accessdata.fda.gov/scripts/opdlisting/oopd/detailedIndex.cfm?cfgridkey=479715).

To our knowledge, this is the first study to compare changes in global gene expression after treatment with calcitriol and one of its low calcemic synthetic analogues by RNA-seq analysis. Using the breast cancer cell line, MCF-7, major overlap was found in the genes differentially regulated by calcitriol and inecalcitol. This finding suggests that the 2 compounds act largely in a similar manner. However, a minority of genes were exclusively regulated by each of the compounds, suggesting that they may not act identically.

In contrast to our study showing uniquely regulated genes by calcitriol and its analogue, inecalcitol, Vanoirbeek and coworkers (Vanoirbeek et al. 2009) using microarray previously reported that calcitriol and the analogue, WY1112 regulated the same set of genes. In contrast to our study that used RNA-seq analysis to identify differential gene expression, this previously published report used microarray. It is generally believed that the newer technique of RNA-seq is considerably more sensitive than microarray in detecting gene expression. Thus, our use of the more sensitive technique may have detected more differentially expressed genes than the microarray used previously. A further reason why calcitriol and inecalciol may induce different genes, in contrast with calcitriol and WY1112, is the possibility that not all vitamin D analogues act similarly. Thus, although calcitriol and WY1112 may regulate the same genes, calcitriol and inecalcitol may not necessarily do so.

Using GO term analysis, genes involved in functions such as bone morphogenesis and ossification were specifically enriched by calcitriol, whereas cell surface signalling and cell communication were specifically enriched after treatment with inecalcitol.

A surprising finding from the GO term analysis, however, was that the term, cellular response to vitamin D, was not enriched after treatment with inecalcitol. The likely explanation for this is that this particular GO category is sparse, containing only 6 genes (TRIM24, BAZ1B, SNAI2, KANK2, VDR and CYP24A1). The calcitriol-regulated gene list contained SNAI2 and CYP24A1, and therefore, reached significance (2/6 vs background). In contrast, the inecalcitol-regulated genes only contained CYP24A1 that failed to reach significance (1/6 vs background). This is a quirk of the GOseq approach (Young et al. 2010) and the small size of this particular GO category.

A key question is whether the induction of any of the unique genes can explain the enhanced cell growth inhibitory potential of inecalcitol compared with calcitriol (Verlinden et al. 2000, Swami et al. 2012). In this context, the SNAI2 gene that was upregulated by calcitriol but not by inecalcitol after RNA-seq analysis was previously reported to suppress VDR expression and decrease calcitriol-mediated cancer cell growth inhibition (Pálmer et al. 2004). Using RT-PCR, we confirmed that SNAI2 was upregulated not only in MCF-7 cells but also in MDA-MB-453, CAMA1 and T47D cells. Unlike our findings with calcitriol, SNAI2 expression was not found to be altered by inecalcitol in any of the 6 cell lines tested. As SNAI2 was previously reported to inhibit calcitriol-mediated cell growth inhibition (Pálmer et al. 2004), this observation of different regulation of SNAI2 by the 2 compounds might at least partly explain the enhanced cell growth suppression mediated by inecalcitol vs calcitriol.

In contrast to SNAI2, MYBPH was found to be upregulated by inecalcitol in all of the 6 cell lines investigated. Calcitriol failed to increase MYBPH in 5 of the six cell lines. As MYBPH has previously been implicated in reducing cancer cell migration and metastasis (Hosono et al. 2012), this finding may provide an explanation for the greater potency of inecalcitol compared to calcitriol in reducing migration (Fig. 4).

Several studies have previously investigated the effects of calcitriol on global gene expression in cell lines (Goeman et al. 2014, Sheng et al. 2015, Simmons et al. 2015). The key conclusion from these reports was that only a small number of genes were found to be consistently regulated in the different studies. Thus, Simmons and coworkers (Simmons et al. 2015) identified only 11 genes that were simultaneously altered in 3 different breast cancer cell lines and only 5 overlapping genes in 4 different datasets, i.e., CYP24A1, CLMN, SERPINB1, IL1R1 and EFTUDE. Of these 5 overlapping genes, 3 were found to be modulated by both calcitriol and inecalcitol in the present study, i.e., CYP24A1, CLMN and EFTUDE.

Likely reasons for the different profile of genes found to be regulated in the different reports include the use of different cell lines, different concentration of calcitriol/analogue used, different lengths of treatment, different sensitivity in technique used to measure gene expression, different cut-off points for defining fold-difference in gene expression and different statistical tools used for the analysis of gene expression.

Although the identity of the calcitriol-regulated genes is variable from report to report, a consistent finding is that the top upregulated gene across all studies is CYP24A1. Indeed, in our study, CYP24A1 was also the top upregulated gene by inecalcitol. However, in our study, the upregulation of CYP24A1 by inecalcitol was approximately 3-fold higher than that by calcitriol, although the concentration of the synthetic analogue used was 500-fold lower than that of calcitriol. CYP24A1 is a member of the cytochrome class 1 P450 family of proteins and is believed to be the key enzyme involved in the inactivation of calcitriol. One of its functions thus appears to be preventing the accumulation of potentially toxic concentrations of calcitriol (Luo et al. 2016). In contrast to the greater potency of inecalcitol vis-à-vis calcitriol in inducing CYP24A1, the extent of the fold-change in the differential regulation of most of the other genes was similar for both compounds.

To identify the likely pathways in which the calcitriol-/inecalcitol-regulated genes were involved, we performed GO analysis. Using the R package known a goseq (Christakos et al. 2016), the top GO term associated with treatment with both inecalcitol and calcitriol was muscle migration. Although muscle has not traditionally been regarded as a classical target tissue for vitamin D, several reports suggest that vitamin D plays a role in both skeletal and smooth muscle cell migration (Rebsamen et al. 2002, Tukaj et al. 2010, Owens et al. 2015). In one of these reports, calcitriol was shown to promote smooth cell migration by upregulating PI3K signalling (Rebsamen et al. 2002). In another, the compound appeared to enhance migration by inducing cytoskeletal reorganisation (Tukaj et al. 2010).

The second most significantly enriched GO term involving both calcitriol and inecalcitol was protein citrullination. Protein citrullination is a post-translational modification of proteins in which arginine residues are converted to citrulline. After a review of the published literature, we were unable to identify any previous report that linked vitamin D with protein citrullination. However, as histone citrullination has previously been associated with gene regulation (Fuhrmann & Thompson 2015, Clancy et al. 2016), vitamin D-mediated citrullination maybe one of the mechanism by which the compound modulates gene expression. To our knowledge, vitamin D has not previously been implicated in protein citrullination.

Similar to the situation in breast cancer cell lines, VDR is expressed in a subset of breast cancers (Berger et al. 1991, Friedrich et al. 2002, Ditsch et al. 2012, Al-Azhri et al. 2017). Published findings, however, are mixed as regards its potential prognostic value in this malignancy (Berger et al. 1991, Friedrich et al. 2002, Ditsch et al. 2012, Al-Azhri et al. 2017). Here, we show that high VDR expression predicted favourable outcome only in patients with luminal A disease, especially if these patients were treated with adjuvant tamoxifen. In contrast, VDR was unrelated to outcome in the total population of patients or in those with luminal B, HER2 or TN disease. Previously, Berger and coworkers (Berger et al. 1991) reported that high VDR levels correlated with a favourable disease-free interval but not with overall survival. Ditsch and coworkers (Ditsch et al. 2012) however, found that high VDR levels predicted good overall survival. On the other hand, Friedrich and coworkers (Friedrich et al. 2002) and Al-Azhri and coworkers (Al-Azhri et al. 2017) concluded that VDR was not prognostic in breast cancer. Unlike our study, those previously reported investigations failed to separate breast cancer patients into the different molecular subgroups. Our finding of VDR being associated with good overall survival, only in patients with luminal A disease may explain the discrepant finding previously reported. A possible reason why patients with high VDR expression have better survival than those with low levels when treated with tamoxifen is that this subgroup of patients has higher levels of ER than those with low levels of VDR (Al-Azhri et al. 2017). Patients with higher ER levels are well known to derive more benefit from tamoxifen than those with low levels of ER.

In conclusion, we show that the low calcaemic-inducing vitamin D analogue, inecalcitol was substantially more potent in blocking cell growth than calcitriol. Inhibition of cell growth was most pronounced in ER-positive cells lines and was dependent on the concentration of cell line VDR levels. Our RNA-seq analysis suggests that there is substantial overlap in the genes differentially regulated by calcitriol and its analogue, inecalcitol. This finding suggests that both compounds act in a largely similar manner. However, as there were some altered genes and GO terms unique to the treatment with each compound, their mode of action is unlikely to be identical. A challenge for future research will be to establish whether any of the uniquely regulated genes can explain the enhanced cell growth inhibitory potency of inecalcitol compared to calcitriol. As inecalcitol has previously been shown to be growth inhibitory in a mouse model of breast cancer (Verlinden et al. 2000) and to have undergone investigations in an early phase clinical trial (Medioni et al. 2014), it should now be considered for evaluation in a clinical trial in breast cancer, especially in ER-positive patients.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/ERC-16-0463.

Declaration of interest

The authors wish to confirm that there are no known conflicts of interest associated with this publication for all co-authors, and there has been no significant financial support for this work that could have influenced its outcome.

Funding

The authors wish to thank the Caroline Foundation, the Cancer Clinical Research Trust (CCRT), Science Foundation Ireland, Strategic Research Cluster Award (08/SRC/B1410) to Molecular Therapeutics for Cancer Ireland (MTCI) and the BREAST-PREDICT (CCRC13GAL) programme of the Irish Cancer Society and funding this work. The opinions, findings and conclusions or recommendations expressed in this article, however, are those of the relevant authors and do not necessarily reflect the views of the funding organisations.

Author contribution statement

A M developed most of the methodology and performed most of the experiments. S M performed the bioinformatics analysis and GO analysis. He also advised with respect to the statistical analysis and wrote the section relating to the RNA-seq analysis. N S assisted with editing manuscript and experimental procedures. R K assisted with the RNA-seq experiment. D O C, N O D, W G and J C contributed reagents and materials and assisted with editing of the manuscript. M J D conceived and directed the study. All authors contributed to the writing of the manuscript, read and approved the final version of the manuscript.

References

  • Al-Azhri J, Zhang Y, Bshara W, Zirpoli G, McCann SE, Khoury T, Morrison CD, Edge SB, Ambrosone CB & Yao S 2017 Tumor expression of vitamin D receptor and breast cancer histopathological characteristics and prognosis. Clinical Cancer Research 23 97103. (doi:10.1158/1078-0432.CCR-16-0075)

    • Search Google Scholar
    • Export Citation
  • Benjamini Y & Hochberg Y 1995 Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series 57 289300. (doi:10.2307/2346101)

    • Search Google Scholar
    • Export Citation
  • Berger U, McClelland RA, Wilson P, Greene GL, Haussler MR, Pike JW, Colston K, Easton D & Coombes RC 1991 Immunocytochemical determination of estrogen receptor, progesterone receptor, and 1,25-dihydroxyvitamin D3 receptor in breast cancer and relationship to prognosis. Cancer Research 51 239244.

    • Search Google Scholar
    • Export Citation
  • Caiazza F, Murray A, Madden SF, Synnott NC, Ryan EJ, O’Donovan N, Crown J & Duffy MJ 2016 Pre-clinical evaluation of the AR inhibitor enzalutamide in triple negative breast cancer cells. Endocrine-Related Cancer 23 323334. (doi:10.1530/ERC-16-0068)

    • Search Google Scholar
    • Export Citation
  • Campbell MJ 2014 Vitamin D and the RNA transcriptome: more than mRNA regulation. Frontiers in Physiology 5 181. (doi:10.3389/fphys.2014.00181)

    • Search Google Scholar
    • Export Citation
  • Chen PT, Hsieh CC, Wu CT, Yen TC, Lin PY, Chen WC & Chen MF 2015 1α,25-Dihydroxyvitamin D3 inhibits esophageal squamous cell carcinoma progression by reducing IL6 signaling. Molecular Cancer Therapeutics 14 13651375. (doi:10.1158/1535-7163.MCT-14-0952)

    • Search Google Scholar
    • Export Citation
  • Christakos S, Dhawan P, Verstuyf A, Verlinden L & Carmeliet G 2016 Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects. Physiological Reviews 96 365408. (doi:10.1152/physrev.00014.2015)

    • Search Google Scholar
    • Export Citation
  • Clancy KW, Weerapana E & Thompson PR 2016 Detection and identification of protein citrullination in complex biological systems. Current Opinion in Chemical Biology 30 16. (doi:10.1016/j.cbpa.2015.10.014)

    • Search Google Scholar
    • Export Citation
  • Ditsch N, Toth B, Mayr D, Lenhard M, Gallwas J, Weissenbacher T, Dannecker C, Friese K & Jeschke U 2012 The association between vitamin D receptor expression and prolonged overall survival in breast cancer. Journal of Histochemistry and Cytochemistry 60 121129. (doi:10.1369/0022155411429155)

    • Search Google Scholar
    • Export Citation
  • Early Breast Cancer Trialists’ Collaborative Group (EBCTCG), Davies C, Godwin J, Gray R, Clarke M, Cutter D, Darby S, McGale P, Pan HC & Taylor C 2011 Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 378 771784. (doi:10.1016/S0140-6736(11)60993-8)

    • Search Google Scholar
    • Export Citation
  • Feldman D, Krishnan AV, Swami S, Giovannucci E & Feldman BJ 2015 The role of vitamin D in reducing cancer risk and progression. Nature Reviews Cancer 14 342357. (doi:10.1038/nrc3691)

    • Search Google Scholar
    • Export Citation
  • Flanagan L, Packman K, Juba B, O’Neill S, Tenniswood M & Welsh J 2003 Efficacy of vitamin D compounds to modulate estrogen receptor negative breast cancer growth and invasion. Journal of Steroid Biochemistry and Molecular Biology 84 181192. (doi:10.1016/S0960-0760(03)00028-1)

    • Search Google Scholar
    • Export Citation
  • Friedrich M, Villena-Heinsen C, Tilgen W, Schmidt W, Reichrat J & Axt-Fliedner R 2002 Vitamin D receptor (VDR) expression is not a prognostic factor in breast cancer. Anticancer Research 22 19191924.

    • Search Google Scholar
    • Export Citation
  • Fuhrmann J & Thompson PR 2015 Protein arginine methylation and citrullination in epigenetic regulation. ACS Chemical Biology 11 654668. (doi:10.1021/acschembio.5b00942)

    • Search Google Scholar
    • Export Citation
  • Goeman F, De Nicola F, D’Onorio De Meo P, Pallocca M, Elmi B, Castrignanò T, Pesole G, Strano S & Blandino G 2014 VDR primary targets by genome-wide transcriptional profiling. Journal of Steroid Biochemistry and Molecular Biology 143 348356. (doi:10.1016/j.jsbmb.2014.03.007)

    • Search Google Scholar
    • Export Citation
  • Haussler MR, Whitfield GK, Kaneko I, Haussler CA, Hsieh D, Hsieh JC & Jurutka PW 2013 Molecular mechanisms of vitamin D action. Calcified Tissue International 92 7798. (doi:10.1007/s00223-012-9619-0)

    • Search Google Scholar
    • Export Citation
  • Hosono Y, Yamaguchi T, Mizutani E, Yanagisawa K, Arima C, Tomida S, Shimada Y, Hiraoka M, Kato S & Yokoi K 2012 MYBPH, a transcriptional target of TTF-1, inhibits ROCK1, and reduces cell motility and metastasis. EMBO Journal 31 481493. (doi:10.1038/emboj.2011.416)

    • Search Google Scholar
    • Export Citation
  • Hughes L, Malone C, Chumsri S, Burger AM & McDonnell S 2008 Characterisation of breast cancer cell lines and establishment of a novel isogenic subclone to study migration, invasion and tumourigenicity. Clinical and Experimental Metastasis 25 549557. (doi:10.1007/s10585-008-9169-z)

    • Search Google Scholar
    • Export Citation
  • Kasiappan R, Sun Y, Lungchukiet P, Quarni W, Zhang X & Bai W 2014 Vitamin D suppresses leptin stimulation of cancer growth through microRNA. Cancer Research 74 61946204. (doi:10.1158/0008-5472.CAN-14-1702)

    • Search Google Scholar
    • Export Citation
  • Leyssens C, Verlinden L & Verstuyf A 2013 Antineoplastic effects of 1,25(OH)2D3 and its analogs in breast, prostate and colorectal cancer. Endocrine-Related Cancer 20 3147. (doi:10.1530/ERC-12-0381)

    • Search Google Scholar
    • Export Citation
  • Liao Y, Smyth GK & Shi W 2013 The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Research 41 e108. (doi:10.1093/nar/gkt214)

    • Search Google Scholar
    • Export Citation
  • Lundqvist J, Yde CW & Lykkesfeldt AE 2014 1α,25-Dihydroxyvitamin D3 inhibits cell growth and NFκB signaling in tamoxifen-resistant breast cancer cells. Steroids 85 3035. (doi:10.1016/j.steroids.2014.04.001)

    • Search Google Scholar
    • Export Citation
  • Lungchukiet P, Sun Y, Kasiappan R, Quarni W, Nicosia SV, Zhang X & Bai W 2014 Suppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D3 and its receptor. Journal of Steroid Biochemistry and Molecular Biology 148 138. (doi:10.1016/j.jsbmb.2014.11.005)

    • Search Google Scholar
    • Export Citation
  • Luo W, Johnson CS & Trump DL 2016 Vitamin D signaling modulators in cancer therapy. Vitamins and Hormones 100 433472. (doi:10.1016/bs.vh.2015.11.004)

    • Search Google Scholar
    • Export Citation
  • Ma Y, Yu WD, Hidalgo AA, Luo W, Delansorne R, Johnson CS & Trump DL 2013 Inecalcitol, an analog of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system. Cell Cycle 12 743752. (doi:10.4161/cc.23846)

    • Search Google Scholar
    • Export Citation
  • Madden SF, Clarke C, Gaule P, Aherne ST, O’Donovan N, Clynes M, Crown J & Gallagher WM 2013 BreastMark: an integrated approach to mining publicly available transcriptomic datasets relating to breast cancer outcome. Breast Cancer Research 15 R52. (doi:10.1186/bcr3444)

    • Search Google Scholar
    • Export Citation
  • McGowan PM, Mullooly M, Caiazza F, Sukor S, Madden SF, Maguire AA, Pierce A, McDermott EW, Crown J & O’Donovan N 2013 ADAM-17: a novel therapeutic target for triple negative breast cancer. Annals of Oncology 24 362369. (doi:10.1093/annonc/mds279)

    • Search Google Scholar
    • Export Citation
  • Medioni J, Deplanque G, Ferrero JM, Maurina T, Rodier JM, Raymond E, Allyon J, Maruani G, Houillier P & Mackenzie S 2014 Phase I safety and pharmacodynamic of inecalcitol, a novel VDR agonist with docetaxel in metastatic castration-resistant prostate cancer patients. Clinical Cancer Research 20 44714477. (doi:10.1158/1078-0432.CCR-13-3247)

    • Search Google Scholar
    • Export Citation
  • Murray A, Synnott N, Crown J, O’Donovan N & Duffy MJ 2015 The vitamin D receptor: a therapeutic target for the treatment of breast cancer? Journal of Clinical Oncology 33 (Supplement) abstract 534. (available at: http://meetinglibrary.asco.org/content/145606-156)

    • Search Google Scholar
    • Export Citation
  • Narvaez CJ, Zinser G & Welsh J 2001 Functions of 1alpha,25-dihydroxyvitamin D(3) in mammary gland: from normal development to breast cancer. Steroids 66 301308. (doi:10.1016/S0039-128X(00)00202-6)

    • Search Google Scholar
    • Export Citation
  • Narvaez CJ, Matthews D, LaPorta E, Simmons KM, Beaudin S & Welsh J 2014 The impact of vitamin D in breast cancer: genomics, pathways, metabolism. Frontiers in Physiology 5 213. (doi:10.3389/fphys.2014.00213)

    • Search Google Scholar
    • Export Citation
  • Okamoto R, Delansorne R, Wakimoto N, Doan NB, Akagi T, Shen M, Ho QH, Said JW & Koeffler HP 2012 Inecalcitol, an analog of 1α,25(OH)(2) D(3), induces growth arrest of androgen-dependent prostate cancer cells. International Journal of Cancer 130 24642473. (doi:10.1002/ijc.26279)

    • Search Google Scholar
    • Export Citation
  • Owens DJ, Sharples AP, Polydorou I, Alwan N, Donovan T, Tang J, Fraser WD, Cooper RG, Morton JP & Stewart C 2015 A systems-based investigation into vitamin D and skeletal muscle repair, regeneration, and hypertrophy. American Journal of Physiology, Endocrinology and Metabolism 309 E1019E1031. (doi:10.1152/ajpendo.00375.2015)

    • Search Google Scholar
    • Export Citation
  • Pálmer HG, Larriba MJ, García JM, Ordóñez-Morán P, Peña C, Peiró S, Puig I, Rodríguez R, de la Fuente R & Bernad A 2004 The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer. Nature Medicine 10 917919. (doi:10.1038/nm1095)

    • Search Google Scholar
    • Export Citation
  • Rebsamen MC, Sun J, Norman AW & Liao JK 2002 1alpha,25-Dihydroxyvitamin D3 induces vascular smooth muscle cell migration via activation of phosphatidylinositol 3-kinase. Circulation Research 91 1724. (doi:10.1161/01.RES.0000025269.60668.0F)

    • Search Google Scholar
    • Export Citation
  • Robinson MD & Oshlack A 2010 A scaling normalization method for differential expression, Genome Biology 11 R25. (doi:10.1186/gb-2010-11-3-r25)

  • Seyednasrollah F, Laiho A & Elo LL 2015 Comparison of software packages for detecting differential expression in RNA-seq studies. Briefings in Bioinformatics 16 5970. (doi:10.1093/bib/bbbib86)

    • Search Google Scholar
    • Export Citation
  • Sheng L, Anderson PH, Turner AG, Pishas KI, Dhatrak DJ, Gill PG, Morris HA & Callen DF 2015 Identification of vitamin D3 target genes in human breast cancer tissue. Journal of Steroid Biochemistry and Molecular Biology 164 9097. (doi:10.1016/j.jsbmb.2015.10.012)

    • Search Google Scholar
    • Export Citation
  • Simboli-Campbell M, Narvaez CJ, Tenniswood M & Welsh J 1996 1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells. Journal of Steroid Biochemistry and Molecular Biology 58 367376. (doi:10.1016/0960-0760(96)00055-6)

    • Search Google Scholar
    • Export Citation
  • Simmons KM, Beaudin SG, Narvaez CJ & Welsh J 2015 Gene signatures of 1,25-dihydroxyvitamin D3 exposure in normal and transformed mammary cells. Journal of Cellular Biochemistry 116 16931711. (doi:10.1002/jcb.25129)

    • Search Google Scholar
    • Export Citation
  • Smyth GK 2004 Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3 article 3. (doi:10.2202/1544-6115.1027)

    • Search Google Scholar
    • Export Citation
  • Swami S, Krishnan AV & Feldman D 2000 1alpha,25-Dihydroxyvitamin D3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells. Clinical Cancer Research 6 33713379.

    • Search Google Scholar
    • Export Citation
  • Swami S, Krishnan AV, Wang JY, Jensen K, Horst R, Albertelli MA & Feldman D 2012 Dietary vitamin D3 and 1,25-dihydroxyvitamin D3 (calcitriol) exhibit equivalent anticancer activity in mouse xenograft models of breast and prostate cancer. Endocrinology 153 25762587. (doi:10.1210/en.2011-1600)

    • Search Google Scholar
    • Export Citation
  • Tukaj C, Trzonkowski P, Pikuła M, Hallmann A & Tukaj S 2010 Increased migratory properties of aortal smooth muscle cells exposed to calcitriol in culture. Journal of Steroid Biochemistry and Molecular Biology 121 208211. (doi:10.1016/j.jsbmb.2010.03.044)

    • Search Google Scholar
    • Export Citation
  • Valenca LB, Sweeney CJ & Pomerantz MM 2015 Sequencing current therapies in the treatment of metastatic prostate cancer. Cancer Treatment Reviews 41 332340. (doi:10.1016/j.ctrv.2015.02.010)

    • Search Google Scholar
    • Export Citation
  • Vanoirbeek E, Eelen G, Verlinden L, Marchal K, Engelen K, De Moor B, Beullens I, Marcelis S, De Clercq P & Bouillon R 2009 Microarray analysis of MCF-7 breast cancer cells treated with 1,25-dihydroxyvitamin D3 or a 17-methyl-D-ring analog. Anticancer Research 29 35853590.

    • Search Google Scholar
    • Export Citation
  • Verlinden L, Verstuyf A, Van Camp M, Marcelis S, Sabbe K, Zhao XY, De Clercq P, Vandewalle M & Bouillon R 2000 Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo. Cancer Research 60 26732679.

    • Search Google Scholar
    • Export Citation
  • Vink-van Wijngaarden T, Pols HA, Buurman CJ, van den Bemd GJ, Dorssers LC, Birkenhäger JC & van Leeuwen JP 1994 Inhibition of breast cancer cell growth by combined treatment with vitamin D3 analogues and tamoxifen. Cancer Research 54 57115717.

    • Search Google Scholar
    • Export Citation
  • Welsh J 1994 Induction of apoptosis in breast cancer cells in response to vitamin D and antiestrogens. Biochemistry and Cell Biology 72 537545. (doi:10.1139/o94-072)

    • Search Google Scholar
    • Export Citation
  • Young MD, Wakefield MJ, Smyth GK & Oshlack A 2010 Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology 11 R14. (doi:10.1186/gb-2010-11-2-r14)

    • Search Google Scholar
    • Export Citation

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    Effect of calcitriol or inecalcitol on cell line growth and relationship between response to calcitriol or inecalcitol treatment and VDR protein levels. (A) Comparative IC50 values for breast cancer cell lines treated with calcitriol or inecalcitol. (B) and (C) scatter plot representing the relationship between IC50 values for calcitriol (B) or inecalcitol (C) and VDR status. Data were analysed using Student t test.

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    Relationship between the response to calcitriol or inecalcitol treatment and the molecular subtype of breast cancer cell line. (A, B, C, D, E and F) Scatter plots representing the relationship between response to treatment with calcitriol (A, C and E) or inecalcitol (B, D and F) and the molecular subtype of breast cancer. Scatter plots representing ER+ and ER− cell lines (A and B), AR+ and AR− cell lines (C and D) or HER2+ or HER2− cell lines (E and F). Data were analysed using the Student’s t test. Data points represent the mean of three independent experiments.

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    Effect of calcitriol or inecalcitol on cell migration. Bar chart representing the percentage cell migration as determined by the Transwell migration, as well as a representative image of Transwell insert (×40) for MCF-7 (A), BT20 (B), Hs578t(i8) (C) or MDA MB 453 cell lines. Cells were treated with either 2 µM calcitriol or 4 nM inecalcitol. Percent migration was determined by quantifying the number of cells on the underside of the insert and normalising to vehicle control. All experiments were carried out in triplicate. Data were analysed using Paired t test. A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0463.

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    Effect of calcitriol or inecalcitol treatment on the expression of SNAI2 and MYBPH. (A) and (B) bar chart representing the fold-change in expression of SNAI2 (A) or MYBPH (B) after either 2 µM calcitriol (grey) or 4 nM inecalcitol (white) treatment of a panel of breast cancer cell lines. Data were analysed using the Student’s t test. All experiments were carried out in triplicate.

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    Effect of VDR expression on disease-free survival in breast cancer. The prognostic value of VDR expression in breast cancer was determined using the BreastMark database (Madden et al. 2013). Kaplan–Meier survival curves for (A) total population (n = 2592, HR = 0.9318, P = 0.290), (B) HER2 subgroup (n = 325, HR = 0.9515, P = 0.7640), (C) basal subgroup (n = 394, HR = 0.8932, P = 0.4347), (D) luminal B subgroup (n = 617, HR = 1.026, P = 0.8149), (E) luminal A subgroup (n = 1035, HR = 0.7393, P = 0.0056) or (F) luminal A treated with adjuvant tamoxifen (n = 273, HR = 0.2726, P = 0.0003). A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0463.