The main regulator of the human tumor suppresser gene p21^(waf1/cip1) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21^(waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions −1400 and −2300 and identified a novel site at position −4500. In addition, we found three VDR-associated promoter regions at positions −2300, −4500 and −6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1α,25(OH)₂D₃ response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1α,25(OH)₂D₃. Moreover, re-ChIP assays confirmed the functionality of the three 1α,25(OH)₂D₃-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21^(waf1/cip1) gene is a primary 1α,25(OH)₂D₃-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.

Purpose: Vitamin D seems to exert a protective effect against common cancers, although this does not correlate with circulating levels of active 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], indicating a more localized activation of vitamin D. The aim of this study was to investigate the significance of this in breast cancer. Experimental Design: Quantitative reverse transcription-PCR analysis of mRNA expression was carried out for the vitamin D–activating enzyme 1α-hydroxylase, the catabolic enzyme 24-hydroxylase, and the vitamin D receptor in 41 tumors and paired nonneoplastic tissue as well as breast cancer cell lines. Immunohistochemistry was used to assess 1α-hydroxylase protein expression, and enzyme assays were used to quantify vitamin D metabolism. Results: Expression of mRNA for 1α-hydroxylase (27-fold; P < 5 × 10⁻¹¹), vitamin D receptor (7-fold; P < 1.5 × 10⁻⁸), and 24-hydroxylase (4-fold; P < 0.02) was higher in breast tumors. 1α-Hydroxylase enzyme activity was also higher in tumors (44.3 ± 11.4 versus 12.4 ± 4.8 fmol/h/mg protein in nonneoplastic tissue; P < 0.05). However, production of inactive 1,24,25-trihydroxyvitamin D₃ was also significantly higher in tumors (84.8 ± 11.7 versus 33.6 ± 8.5 fmol/h/mg protein; P < 0.01). Antisense inhibition of 24-hydroxylase in vitro increased antiproliferative responses to 1,25(OH)₂D₃. Conclusion: These data indicate that the vitamin D–activating enzyme 1α-hydroxylase is up-regulated in breast tumors. However, dysregulated expression of 24-hydroxylase seems to abrogate the effects of local 1,25(OH)₂D₃ production in tumors by catalyzing catabolism to less active vitamin D metabolites. The enzymes involved in autocrine metabolism of vitamin D in breast tissue may therefore provide important targets for both the prevention and treatment of breast cancer.

KDM6B/JMJD3 is a histone H3 lysine demethylase with an important gene regulatory role in development and physiology. Here, we show that human JMJD3 expression is induced by the active vitamin D metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and that JMJD3 modulates the gene regulatory action of this hormone. 1,25(OH)₂D₃ activates the JMJD3 gene promoter and increases the level of JMJD3 RNA in human cancer cells. JMJD3 upregulation was strictly dependent on vitamin D receptor (VDR) expression and was abolished by cycloheximide. In SW480-ADH colon cancer cells, JMJD3 knockdown or expression of an inactive mutant JMJD3 fragment decreased the induction by 1,25(OH)₂D₃ of several target genes and of an epithelial adhesive phenotype. Moreover, JMJD3 knockdown upregulated the epithelial-to-mesenchymal transition inducers SNAIL1 and ZEB1 and the mesenchymal markers fibronectin and LEF1, while it downregulated the epithelial proteins E-cadherin, Claudin-1 and Claudin-7. Additionally, JMJD3 knockdown abolished the nuclear export of β-catenin and the inhibition of β-catenin transcriptional activity caused by 1,25(OH)₂D₃. Importantly, the expression of JMJD3 correlated directly with that of VDR and inversely with that of SNAI1 in a series of 96 human colon tumours. Our results indicate for the first time that an epigenetic gene coding for a histone demethylase such as JMJD3 is a VDR co-target that partially mediates the effects of 1,25(OH)₂D₃ on human colon.

We have synthesized and studied the ability of a series of seven novel 1α,25(OH)₂ vitamin D₃ analogues to inhibit clonal growth of prostate cancer cells (LNCaP, PC-3 and DU-145). Addition of double and triple bonds to the C/D ring (C-16) and side chain (C-22 and C-23) as well as lengthening of the side chain were important for enhanced activity against LNCaP and PC-3. Reorientation of the side chain in the 20-epi configuration resulted in analogues that were extremely potent only against LNCaP (ED₅₀ ≈ 5 × 10⁻¹¹ m). Compounds with six fluorines on the end of the side chain were very active against both PC-3 and LNCaP (ED₅₀ ≈ 2 × 10⁻⁸ m). DU-145 cells were relatively resistant to compounds with all of these modifications, but removal of C-19 (e.g. 1,25(OH)₂-16-ene-23-yne-26,27-F₆-19-nor-D₃) resulted in an analogue that was inhibitory against all three prostate cell lines. Further analysis showed that pulse exposure (3 days, 10⁻⁷ m) to this analogue was enough to inhibit clonal growth of PC-3 cells by 50%. The same exposure also induced cell cycle arrest of all three cell lines, accompanied by upregulated protein expression of the cyclin-dependent kinase inhibitor (CDKI) known as p21^waf1 in all three cell lines, and the CDKI known as p27^kip1 in LNCaP cells. Associated with upregulation of these CDKIs, partial differentiation occurred as measured by increased expression of both prostate-specific antigen by LNCaP cells and E-cadherin, a cell adhesion protein that may act as a putative tumour suppressor (LNCaP and PC-3 cells). In summary, this is the first report of a potent series of 19-nor-vitamin D₃ analogues with the ability to inhibit proliferation of LNCaP, PC-3 and DU-145 prostate cancer cell lines. These compounds may mediate their potent anti-proliferative activities through a cell cycle arrest pathway.