The primary goal of this work was to evaluate the transcriptional responses of breast cancer samples to physiologically relevant concentrations of 1,25(OH)2D3, using a culture model that retains features of intact tumors, such as stromal-epithelial interactions. Microarray analysis identified nine genes that were significantly altered within 24 h of exposure to 1,25(OH)2D3 0.5nM, a concentration that is physiologically achievable in patients. Of these, the vitamin D target gene CYP24A1(which codes a cytochrome P450 enzyme, that hydroxylates 25(OH)D3 and 1,25(OH)2D3 to less active forms 24,25(OH)2D3 and 1,24,25(OH)3D3) was induced over 7-fold in microarray analysis and was validated in another set of tumor samples, clearly indicating activation of VDR signaling. Additional evidence for activation of the VDR pathway in this dataset was obtained by GSEA, which indicated a trend towards the enrichment of genes sharing DR3 binding sites, a consensus motif for VDR.
Comparison of microarray data from tumor slices cultured with 0.5nM vs. 100nM 1,25(OH)2D3 indicated a clear concentration effect, as the number of differentially expressed transcripts increased from nine at 0.5nM to 186 at 100nM (20 fold increment). Induction of CYP24A1 increased from 7-fold (at 0.5 nM) to 70-fold (at 100nM) - a 10 fold enhancement. In both datasets, the majority of genes (approximately 75%) were up-regulated rather than down-regulated by 1,25(OH)2D3, consistent with other array data from established cell lines cultured with high dose 1,25(OH)2D3
In addition to CYP24A1, five other genes were commonly up-regulated in tumor slices exposed to both low and high concentrations of 1,25(OH)2D3: DPP4, KCKN3, EFTUD1, TKTL1 and CA2. All, except TKTL1 (transketolase-like 1) have been previously identified as VDR target genes in various model systems. DPP4 (dipeptidyl-peptidase 4, also called CD26) was up-regulated in artery smooth muscle cells exposed to 1,25(OH)2D3 and its overexpression in distinct cell types (melanocytes, non-small cell lung, prostate and neuroblastoma cells) triggered anti-tumorigenic effects including cell growth arrest, inhibition of cell migration and increased apoptosis . KCNK3 (potassium channel, subfamily K, member) was induced by 1,25(OH)2D3 in artery smooth muscle cells, and EFTUD1 (elongation factor Tu GTP binding domain containing 1) in oral squamous carcinoma, breast cancer associated fibroblasts, immortalized prostate cells and lymphoblastoid cell lines [40, 43–46]. CA2 (carbonic anhydrase II) mRNA appeared to be directly induced by 1,25(OH)2D3 in myelomonocytic cell lines but indirectly regulated in osteoclast progenitors, where the physical communication with stromal cells seems to be required [48, 49]. CYP26B1 (cytochrome P450, family 26, subfamily b, polypeptide 1) which was up-regulated in samples treated with 1,25(OH)2D3 0.5nM, was previously identified as a vitamin D induced gene in immortalized non-transformed prostate epithelial and oral squamous carcinoma cell lines, and in silico analysis has tentatively identified a VDR binding site at this genomic region [43, 44].
Other authors have analyzed physiological concentration effects of vitamin D using animal models. Vitamin D supplemented diet as well as calcitriol injections were shown to stimulate the VDR pathway, mildly increasing CYP24A1 expression (x2) in MCF-7 xenografts in immunocompromised mice . Interestingly, vitamin D transcriptional effects may not overlap in tumor specimens and non-transformed mammary glands in the MMTV-neu transgenic mouse model of breast cancer, fed a high vitamin D diet . Comparison between cancer and normal cells is an interesting issue, as vitamin D potential effects in cancer prevention have also been claimed. In accordance with the previous work , differences in transcriptional targets were also described for breast cancer associated fibroblasts (CAF) and normal adjacent fibroblasts (NAF) exposed to 1,25(OH)2D3 in a supra-physiological concentration. Among up-regulated genes 45.7% were commonly modulated in CAFs and NAFs, however, 36.4% were exclusively up-regulated in NAFs and 17.4% exclusively up-regulated in CAFs . In addition, looking at overlapping genes in the Venn diagram of vitamin D up-regulated transcripts in six works [40, 43–46], only seven intersections were found in non-cancer cells: AKR1B1, CRIP1, FZD8, MREG (in immortalized prostate cells and NAF), BCAT1, GCLC (in coronary artery smooth muscle cells and NAFs) and PRR6 (in immortalized prostate cells and coronary artery smooth muscle cells). Furthermore, it was reported that vitamin D response is blunted in transformed HME normal mammary cells as compared with parental normal cells . The last works evaluating vitamin D effects in normal cells however, were performed using supra-physiological concentrations of 1,25(OH)2D3 (10-100nM) or analogs and the role of physiological concentrations of the hormone in normal cells is not fully established.
At 100nM, 1,25(OH)2D3 exerted more extensive transcriptional effects, and at least 40 of the induced genes in breast cancer organotypic culture have already been reported as up regulated by the hormone, such as ALCAM, ARRDC4, BMP2, BMP6, CA2, CD14, CLIC6, CILP, CLMN, CYP19A1, DCLDB1, EFTUD1, EHBP1, FAM20C, FOXF1, FRAS1, GOS2, GRK5, HBGEF, HSMPP8, IL1RL1, KCNK3, KIAA0500, PKD2, RGNEF, SEMA6D, SERPINB1, SLC1A1, THBD, TIMP1, TRIM56[40, 43–46]. However, co-aggregation of paired samples (treated and untreated) upon cluster analysis suggests that an individual dominant transcriptional profile was maintained, regardless of treatment. These results were not unexpected, as a high degree of transcriptional similarity was also demonstrated for matched pre and post-neoadjuvant chemotherapy, even though the chemotherapy exerts a more pronounced acute cellular effect than hormonal treatments [53–55].
Some of the genes induced by 100nM 1,25(OH)2D3 concentration are involved in TGF beta signaling pathway, in accordance with other authors [56, 57]. Other genes are involved in regulation of leukocyte mediated immunity and positive regulation of alpha-beta T cell activation, including CD14, which encodes a receptor to bacterial lipopolysaccharide, as previously reported in a variety of cells as mononuclear phagocytes, normal human epidermal keratinocytes, oral squamous carcinoma, immortalized non-transformed prostate epithelial cell lines and malignant breast cells [43, 56, 58].
The present tumor slice model represents a heterogeneous combination of epithelial and stromal cells, in which the complex array of reciprocal interactions taking place in the tumor microenvironment, including cell-cell contacts and a variety of secreted factors, might modulate the overall response to 1,25(OH)2D3. Hence, after evaluating the hormone effects in tumor slices, the effects of 1,25(OH)2D3 0.5nM in defined populations of cancer associated fibroblasts and epithelial cells were compared. This data indicated that even though CYP24A1 was induced in both fibroblasts and epithelial cells, CD14, CA2, and IL1RL1 were primarily induced in epithelial cells. There was also a trend towards up-regulation of CA2, DPP4 and IL1RL1 in cancer associated fibroblasts.
One major strengthen of this work was the comparison of achievable versus supra-physiological concentrations of 1,25(OH)2D3 in breast cancer slices, a model that preserves the epithelial-mesenchimal interactionss, indicating that effects are much less intense in near physiological concentrations. However, a weakness of this work was the small number of samples used in microarray experiments. These effects however, were later confirmed in a larger number of tumor samples and cell lines, using RT-PCR, even though they were more difficult to detect at the protein level, in face of the discrete changes induced by 0.5nM 1,25(OH)2D3.