Vitamin D signalling pathways in cancer: potential for anticancer

The most widely accepted physiological role of vitamin D, which is mediated primarily by 1α,25(OH)2D3 (also known as calcitriol), the most active product of vitamin D synthesis, is in the physiological regulation of Ca2+ and Pi transport and bone mineralization1. The importance of this role is demonstrated by studies with knockout mice deficient in key members of the vitamin D metabolic pathway, such as 25-hydroxyvitamin D3-1α-hydroxylase (1α-OHase; encoded by Cyp27b1), an enzyme that gener-ates 1α,25(OH)2D3, and 25-hydroxyvitamin D 24-hydrox-ylase (24-OHase; encoded by Cyp24a1), the enzyme that degrades 1α,25(OH)2D3 (catabolism), and the vitamin D receptor (Vdr), which binds 1α,25(OH)2D3 to affect target gene transcription2–6 (TABLEs 1,2). Loss of these genes in mice led to phenotypes with abnormal bone morphol-ogy. However, recent observations indicate a much broader range of action for 1α,25(OH)2D3, including the regulation of differentiation, proliferation and apoptosis. Furthermore, altered expression and function of proteins crucial in vitamin D synthesis and catabolism have been observed in many tumour types (TABLE 3). Interestingly, Vdr–/– mice show hyperproliferation and increased mitotic activity in the descending colon, suggesting a role for 1α,25(OH)2D3-mediated signalling in tumour suppres-sion7. Zinser and colleagues8–10 showed that Vdr ablation in the mouse increased chemical carcinogenesis in mam-mary, epidermis and lymphoid tissues but not in ovary, uterus, lung or liver (TABLE 2). However, mice deficient in key members of the vitamin D synthesis and catabolic pathways do not develop spontaneous cancer2,3,11.

The seminal finding by Garland and Garland12 of higher mortality rates from colon cancer in the northeast and lower rates in the south, southwest and west in the United States led to the important concept that exposure to ultraviolet B or sunlight, which leads to vitamin D synthesis, can reduce the risk of colorectal cancer. Several epidemiological observations have shown an associa-tion between low serum 25(OH)D3 levels (the accepted measure of vitamin D body stores) and increased risk for colorectal13, breast14 and prostate15 cancers. There are many epidemiological studies that have sought to determine associations between vitamin D status and the risk and mortality rates of a number of cancers16–19. Giovannucci and colleagues16 recently performed an extensive analysis that combined major determinants of vitamin D status on cancer risk and mortality with 51,529 men that were enrolled in the Health Professionals Follow-up Study (HPFS). Individuals were prospectively followed for almost 20 years; diet, exercise and lifestyle characteristics were analysed and health outcomes, including cancer and cancer-related deaths, were assessed. Giovannucci et al. developed a model to pre-dict 25(OH)D3 levels based on the relationship between dietary and supplementary vitamin D, physical activity, body mass index and sunlight exposure (a source of vita-min D). The model was applied to 47,800 individuals in the HPFS, and the analysis indicated a strong association between low levels of predicted 25(OH)D3 and increased cancer incidence and cancer-related mortality, particu-larly for cancers of the digestive system16. Furthermore,

Departments of Pharmacology and Therapeutics* and Medicine‡, Roswell Park Cancer Institute, Buffalo, New York, USA.Correspondence to C.S.J. e-mail: [email protected]:10.1038/nrc2196

Vitamin D signalling pathways in cancer: potential for anticancer therapeuticsKristin K. Deeb*, Donald L. Trump‡ and Candace S. Johnson*

Abstract | Epidemiological studies indicate that vitamin D insufficiency could have an aetiological role in various human cancers. Preclinical research indicates that the active metabolite of vitamin D, 1α,25(OH)2D3, also known as calcitriol, or vitamin D analogues might have potential as anticancer agents because their administration has antiproliferative effects, can activate apoptotic pathways and inhibit angiogenesis. In addition, 1α,25(OH)2D3 potentiates the anticancer effects of many cytotoxic and antiproliferative anticancer agents. Here, we outline the epidemiological, preclinical and clinical studies that support the development of 1α,25(OH)2D3 and vitamin D analogues as preventative and therapeutic anticancer agents.

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684 | SEPTEMBER 2007 | vOLUME 7 www.nature.com/reviews/cancer

© 2007 Nature Publishing Group

http://www.cancer.gov/cancertopics/types/colon-and-rectal

http://www.cancer.gov/cancertopics/types/breast

http://www.cancer.gov/cancertopics/types/prostate

mailto:[email protected]

mailto:[email protected]

Secosteroid hormonesMolecules that are very similar in structure to steroids but with a ‘broken’ ring; two of the B‑ring carbon atoms (C‑9 and 10) of the four steroid rings are not joined.

AutocrineA substance secreted by a cell that acts on the surface receptors of the same cell.

ParacrineA substance secreted by a cell that acts on adjacent cells.

Giovannucci et al. reported that an increase of 25 nmol per L in predicted 25(OH)D3 level is associated with a 29% reduction in cancer-related mortality and a 17% reduction in cancer incidence, suggesting that high 25(OH)D3 levels might be associated with a decreased risk of some cancers16. A meta-analysis of case–control and cohort studies found that individuals with ≥ 33 ng per ml (82 nmol per L) 25(OH)D3 had a 50% lower incidence of colorectal cancer20. Additionally, patients with early stage non-small-cell lung cancer with high 25(OH)D3 levels and high vitamin D intake at the time of diagnosis and initiation of treatment had improved overall and recur-rence-free survival21. Therefore, these data suggest that low levels of 25(OH)D3 are an important risk factor for cancer incidence.

Synthesis and catabolism of vitamin D1α,25(OH)2D3 is synthesized from vitamin D in a highly regulated multistep process (FIG. 1). The first step in vitamin D synthesis is the formation of vita-min D3 in the skin through the action of ultraviolet irradiation; vitamin D3 can also be taken in the diet but in North America and Europe dietary vitamin D3 intake is a minor component of vitamin D3 acquisi-tion because dairy products, eggs, fish and fortified foods contain only small quantities of vitamin D22. Decreased sun exposure further limits vitamin D synthesis.

vitamin D3 (cholecalciferol) is hydroxylated by liver mitochondrial and microsomal 25-hydroxylases (25-OHase)23, encoded by the gene CYP27A1. The

resultant 25-hydroxycholecalciferol (25(OH)D3) is 1α-hydroxylated in the kidney by mitochondrial 1α-hydroxylase (1α-OHase; encoded by the gene CYP27B1), this yields the hormonally active secosteroid 1α,25(OH)2D3 (calcitriol)23. 24-hydroxylation of 25(OH)D3 and 1α,25(OH)2D3 by the cytochrome P450 enzyme 25-hydroxyvitamin D 24-hydroxylase (24-OHase; encoded by the gene CYP24A1), to the metabolites 24,25(OH)2D3 and 1α,24,25(OH)2D3, respectively, is the rate-limiting step for 25(OH)D3 and 1α ,25(OH)2D3 catabolism23. Additionally, 1α,25(OH)2D3 concentrations are feedback regulated: an increase in 24,25(OH)2D3 induces the synthesis of 1α,25(OH)2D3; whereas Ca2+, Pi and 1α,25(OH)2D3 itself suppress 1α,25(OH)2D3 synthesis23–26. CYP27B1 (which encodes 1α-OHase) expression is induced by parathyroid hormone (PTH)25 and repressed by 1α,25(OH)2D3

24,27. Furthermore, CYP24A1 is strongly induced by 1α,25(OH)2D3 to produce the less active vitamin D metabolites 1α,24,25(OH)2D3 and 24,25(OH)2D3

23.There are instances of tissue-specific regulation

of the vitamin D synthetic enzymes. 1α,25(OH)2D3 functions in an autocrine and paracrine manner to modulate vitamin D function and signalling. 1α-OHase is expressed at extra-renal sites such as nor-mal colon, brain, placenta, pancreas, lymph nodes and skin28, allowing local conversion of 25(OH)D3 to 1α,25(OH)2D3. Importantly, increased CYP27B1 expression is observed in breast29 and prostate30 can-cers and during early colon tumour progression in well-to-moderately differentiated states, but decreased in poorly differentiated colon carcinomas31–33. Increased expression of CYP27B1 in cancer tis-sues could provide local conversion of 25(OH)D3 to 1α,25(OH)2D3, and may support the notion that 25(OH)D3 and 1α,25(OH)2D3 might have a role in the chemoprevention of these cancers. However, CYP24A1 (encoding 24-OHase) mRNA expression is upregu-lated in tumours, and may counteract 1α,25(OH)2D3 antiproliferative activity, presumably by decreasing 1α,25(OH)2D3 levels34,35 (TABLE 3). Cross et al.35 have demonstrated that the upregulation of CYP24A1 and downregulation of CYP27B1 can occur in high-grade colon carcinomas. The chromosomal region 20q13.2, containing the CYP24A1 gene, is amplified in human breast tumours36, and CYP24A1 mRNA expression is upregulated in samples from human lung, colon and ovarian tumours, suggesting that 1α,25(OH)2D3 levels would be reduced in these cases35,37,38 (TABLE 3). This suggests that inhibition of CYP24A1 expression and activity is essential for prevention to be effective. Small-molecule inhibitors with varying specificity for 24-OHase39–42 render tumour cells more sensitive to the action of 1α,25(OH)2D3 and its analogues. Consistent with the epidemiological studies discussed above, these findings indicate that 1α,25(OH)2D3 catabolism could modulate tumour growth in some tissues, indicat-ing the potential for the development of 24-OHase inhibitors as cancer preventative and/or anticancer therapeutic agents.

At a glance

•Epidemiological studies point to a relationship between vitamin D deficiency and cancer risk.

•Alterations in vitamin D receptor expression, and in the synthesis (25-hydroxylase and 1α-hydroxylase) and catabolism (24-hydroxylase) of vitamin D metabolites are involved in the growth regulation of tumours; thus, compromising 1α,25(OH)2D3 (also known as calcitriol; the active metabolite of vitamin D signalling) sensitivity and 1α,25(OH)2D3 signalling.

•The antiproliferative effects of 1α,25(OH)2D3 have been demonstrated in various tumour types, as determined by preclinical trials.

•The anti-tumour effects of 1α,25(OH)2D3 involve mechanisms that are associated with G0/G1 arrest, differentiation, induction of apoptosis and modulating different signalling pathways in tumour cells, as well as inhibiting tumour angiogenesis.

•Glucocorticoids potentiate the anti-tumour effects of 1α,25(OH)2D3 and decrease 1α,25(OH)2D3-induced hypercalcemia. 1α,25(OH)2D3 also potentiates the anti-tumour effects of many chemotherapeutic agents such as platinum analogues, taxanes and DNA-intercalating agents.

•Given that the major vitamin D catabolizing enzyme, CYP24A1 (24-hydroxylase), is often amplified and overexpressed in tumour cells, agents that inhibit this enzyme can potentiate 1α,25(OH)2D3 anti-tumour effects.

•Preclinical data indicate that maximal anti-tumour effects are seen with pharmacological doses of 1α,25(OH)2D3, and can be safely achieved in animals using a high-dose, intermittent schedule of administration. Some clinical trial data indicates that 1α,25(OH)2D3 is well-tolerated in cancer patients within a proper dosing schedule.

•Data support the hypothesis that vitamin D compounds may have an important role in cancer therapy and prevention, and merit further investigation.

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NATURE REvIEwS | cancer vOLUME 7 | SEPTEMBER 2007 | 685


http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1593&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum



1α,25(OH)2D3-mediated transcription of target genes. 1α,25(OH)2D3 exerts transcriptional activation and repression of target genes by binding to the vDR (BOX 1). The vDR is a member of the steroid hormone receptor superfamily and regulates gene expression in a ligand-dependent manner43 (FIG. 2). Interestingly, N-terminal vDR variants show tissue-specific expression44,45 that might also contribute to the differential specificity of 1α,25(OH)2D3-mediated regulation. 1α,25(OH)2D3–vDR-dependent transcriptional activity is modulated through synergistic ligand-binding and dimerization with retinoic X receptor (RXR). The activated 1α,25(OH)2D3–vDR–RXR com-plex specifically binds to vitamin D response elements (vDREs), composed of two hexanucleotide repeats inter-spaced by varying numbers of nucleotides (for example, GGTCCA-NNN-GGTCCA, where N is any nucleotide; this is denoted DR3), in the promoter regions of target genes46. For transcriptional activation, vDR occupies the 3′ half-site whereas RXR binds the 5′ half-site of vDRE47.

Co-factor proteins also have the ability to modulate vDR-mediated gene expression; these proteins possess intrinsic chromatin-modifying enzymatic activities, act as a platform for the recruitment of chromatin-modifying proteins and recruit basal transcription factors to the pro-moters23. 1α,25(OH)2D3 binding induces phosphoryla-tion and conformational changes in vDR, which causes the release of co-repressors (such as nuclear receptor co-repressors (NCoRs) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT)–histone deacetylase (HDAC) complex) that maintain chromatin

in a transcriptionally repressed state48. Conformational change also repositions the vDR activation function 2 (AF2) domains to bind to stimulatory coactivators, consisting of the steroid receptor coactivators (SRCs), nuclear coactivator 62 kDa–SKI-interacting protein (NCoA62–SKIP) and the chromatin modifiers, CREB binding protein (CBP)–p300 and PBAF (polybromo- and SwI-2-related gene 1 associated factor), which acetylates histones in the nucleosomes to unravel DNA for transcrip-tion49. Once the chromatin is de-repressed, the vitamin D receptor-interacting proteins (DRIPs) form a complex that binds to the AF2 domain of vDR and interacts with the transcription machinery, such as TF2B (transcription factor 2B) and RNA polymerase II, and initiates transcrip-tion (for a review see REF. 23). Recently, it has been shown that epigenetic regulation of VDR through increased expression of NCoR1 and SMRT repress vDR-mediated signalling in prostate50 and breast cancer51 cell lines, and may have a role in mediating the antiproliferative effects of 1α,25(OH)2D3 in these tissues.

The mechanism by which 1α,25(OH)2D3 represses gene expression through the binding of vDR to negative vDREs (DR3-type), placing vDR on the 5′ half-site of the vDRE52,53, such as is the case with human PTH, may involve interference with transcriptional machinery but is less understood. Recently, transcriptional repression by 1α,25(OH)2D3 has been further elucidated for the human CYP27B1 (REFs 27,54,55) and PTH56 genes. The vDR–RXR heterodimer represses gene transcription in a 1α,25(OH)2D3-dependent manner through E-box-type

Table 1 | Mouse models of vitamin D metabolic enzymes and receptor signalling

Genetic modification Mouse phenotype cancer phenotype refs

Cyp27b1–/– (which encodes 1α-OHase)

Pseudo-vitamin D deficiency rickets (PDDR); decreased serum Ca2+ and Pi; secondary hyperthyroidism; undetectable 1α,25(OH)2D3 (calcitriol) levels; disorganized growth plate structure and osteomalacia. In addition: infertile females; uterine hypoplasia; decreased ovarian size; compromised folliculogenesis; reduction in CD4+ and CD8+ peripheral T lymphocyte

None 2,3

Cyp24a1–/– (24-OHase) Lethal hypercalcemia; impaired intramembranous bone mineralization None 11

Vdr–/– (VDR signalling) Vitamin D deficiency rickets type II (VDDR II) and osteomalacia; alopecia, hypocalcemia, hyperparathyroidism; impaired bone formation, growth retardation; female infertility, uterine hypoplasia, impaired folliculogenesis

Hyperproliferation of descending colon (increased PCNA positivity and cyclin D1 expression)

4,5,6,7

PCNA, proliferating cell nuclear antigen.

Table 2 | Vdr knockout mice and carcinogenesis

Oncogene/carcinogen

Tissue cancer phenotype ref

MPA plus DMBA carcinogens

Skin 40% sebaceous, 25% squamous and 15% follicular papillomas compared with WT littermates; other infrequent lesions include basal cell carcinoma and haemangioma

9

Mammary Higher incidence of alveolar and ductal hyperplasias in Vdr–/– mice compared with WT mice; development of palpable mammary tumours was not altered by Vdr ablation

8

Lymph nodes and/or thymus

Lymphoblastic and thymic lymphoma higher in Vdr–/– (27%) compared with WT mice (11%)

8

Vdr–/– ;neu oncogene Mammary Decreased survival of Vdr–/–; neu mice compared with their Vdr+/+; neu and Vdr+/–; neu littermates; increased development of mammary tumours driven by the neu oncogene

10

DMBA, 7,12-dimethylbenzanthracene; MPA, medroxyprogesterone acetate; WT, wild-type.

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negative vDREs, comprised of a CANNTG-like motif in the promoter regions of the CYP27B1 (REFs 27,54,55) and PTH56 genes, which are distinct from the DR3-type response elements. vDR-interacting repressor (vDIR), when bound to E-box-type elements, induces the transcriptional activation of CYP27B154. However, the binding of 1α,25(OH)2D3 to vDR causes vDR to interact with vDIR. 1α,25(OH)2D3-induced association between vDR and vDIR induces dissociation of the histone acetyltransferase (HAT) co-activator and recruit-ment of HDAC co-repressor for 1α,25(OH)2D3-induced transrepression of CYP27B1 gene expression54. In addi-tion, williams syndrome transcription factor (wSTF) potentiates 1α,25(OH)2D3-induced transrepression by vDR of the CYP27B1 gene promoter by facilitating the association between wINAC, a multifunctional, ATP-dependent chromatin-remodelling complex, and chro-matin55. This transrepression mechanism is an important biological function of vDR to allow negative-feedback

control of 1α,25(OH)2D3 biosynthesis55, as well as negative regulation of other genes with nvDREs in their promot-ers. Furthermore, Kim et al.57 demonstrated that not only is histone deacetylation crucial for chromatin structure remodelling in suppression of the CYP27B1 gene, but that transrepression by vDR requires DNA methylation of the CYP27B1 gene promoter, suggesting complicated epigenetic modifications for transcriptional regulation of the CYP27B1 gene. Epigenetic regulation of CYP27B1 and CYP24A1 has been previously reported for the PNT-2 human normal prostate cells and DU-145 prostate cancer cell line58. Histone methylation and demethylation are cru-cial events that impose ligand- and signal-dependent gene activation by nuclear receptors and prevent the recruit-ment of unliganded nuclear receptors and transcription factors from binding to their target promoters and causing constitutive gene activation59.

Examples of genes with DR3-type response elements that are transcriptionally activated by 1α,25(OH)2D3

Table 3 | Expression of molecules that function in vitamin D metabolism and signalling in human cancers.

Protein (gene)

altered expression observed in human cancer tissues

Prognostic or histological observations

Types of cancer

Vitamin D metabolic enzymes

25-OHase (CYP27A1)

Increased mRNA NA Breast34, cervical34 and ovarian cancer34, HCC172

1α-OHase (CYP27B1)

Increased mRNA NA Basal cell carcinoma173, breast34,174, cervical34 and ovarian cancer34

Increased mRNA Moderately differentiated Colon cancer31,35,175

Decreased mRNA Poorly differentiated Colon cancer35

Splice variants (Hyd-V5, -V6, -V7 and -V8)

NA Glioblastoma multiforme176, melanoma177, cervical cancer177

Immunoreactivity NA Pancreatic132, breast132 and colon cancer 33, renal cell carcinoma132

Increased immunoreactivity Moderately differentiated Colon cancer33,35

Decreased immunoreactivity Poorly differentiated Colon cancer33,35

24-OHase (CYP24A1)

Amplified at 20q13.2 locus NA Gastric adenocarcinoma37, breast cancer36

Increased mRNA NA Basal cell carcinoma173, SCC (cutaneous)178, lung38,42, breast34,36, colon35,38, cervical34 and ovarian cancer34,38

Increased mRNA Poor prognosis Oesophageal cancer179

Decreased mRNA NA Breast cancer38

Increased mRNA and activity Poorly differentiated Colon cancer32

Increased protein NA Lung cancer (NSCLC)42

Vitamin D receptor

VDR (VDR)

Increased mRNA NA Basal cell carcinoma173, SCC (cutaneous)178, colon cancer31

Decreased mRNA Poorly differentiated Colon cancer31

Increased immunoreactivity NA Breast34, cervical34 and ovarian cancer34

Increased, predominantly cytoplasmic

Well differentiated Colon cancer166

Decreased immunoreactivity Moderately and poorly differentiated

Colon cancer166

Poorly differentiated Colon cancer35

HCC, hepatocellular carcinoma; NA, non applicable; NSCLC, non-small cell lung carcinoma; SCC, squamous cell carcinoma.

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Nature Reviews | Cancer

UV-B

Skin

Circulation

Liver

Kidney

Intestine

D3

Pre-D3

7-dehydrocholesterol D3

D3

D3

DBP

D3

25(OH)D3

25-OHase

24-OHase

24,25(OH)2D3

1α,24,25(OH)2D3

Excretion

1α-OHase

Pi, Ca2+ andother factors

+/–

24-OHase

IntestineIncreases absorption of Ca2+ and Pi

BoneIncreases bonemineralization

Immune cellsInducesdifferentiation

Tumourmicroenvironment• Inhibits proliferation• Induces differentiation• Inhibits angiogenesis

Parathyroidglands

PTH +

Dietary sources of vitamin D

1α,25(OH)2D3

consist of CYP24A1 (REF. 60) (encoding 24-OHase), BGLAP61 (osteocalcin; expressed in bone osteoblasts), and CDKN1A62 (which encodes the cyclin depend-ent kinase (CDK) inhibitor p21). Those repressed by 1α,25(OH)2D3 include PTH53. Although vDREs are traditionally thought to occur in the promoter regions of the target genes, a DR3-type vDRE was recently identified in exon 4 of the growth arrest and DNA-damage-inducible (GADD45) gene63. 1α,25(OH)2D3-mediated repression or activation of many proto-oncogenes or tumour-suppressor genes is described in normal and tumour tissues62,64–67; however, only a few such genes contain vDREs in the promoter regions and are under the direct transcrip-tional control of 1α,25(OH)2D3, such as CDKN1A62 and CCNC (which encodes cyclin C, containing a DR4-type vDRE)65. This suggests that 1α,25(OH)2D3 exerts many of its effects indirectly by modulating signalling cascades or by unknown nongenomic mechanisms (FIGs 2,3).

Nongenomic action of 1α,25(OH)2D3. Nongenomic actions mediated by 1α,25(OH)2D3 are rapid and not dependent on transcription. However, nongenomic signalling may indirectly affect transcription through cross-talk with other signalling pathways68,69. Although there is no agreement on how the nongenomic actions are initiated, data suggest that these effects begin at the plasma membrane and involve a non-classical membrane receptor (memvDR; FIG. 2) described in intestinal caveo-lae70, and a 1α,25(OH)2D3-membrane-associated rapid-response steroid binding protein (1α,25D3-MARRS) isolated from chick intestinal basal-lateral membrane71.

The most well-described nongenomic effect of 1α,25(OH)2D3 is the rapid intestinal absorption of Ca2+

(REF. 72). Binding of 1α,25(OH)2D3 to the proposed mem-brane receptor can result in the activation of numerous signalling cascades68,69 (FIG. 2). Activation of these signalling cascades, such as protein kinase C (PKC), can result in the rapid opening of voltage-gated Ca2+ channels and an increase in intracellular Ca2+ (REF. 73), which may subsequently

Figure 1 | Vitamin D metabolism. Photochemical synthesis of vitamin D3 (cholecalciferol, D3) occurs cutaneously where pro-vitamin D3 (7-dehydrocholesterol) is converted to pre-vitamin D3 (pre-D3) in response to ultraviolet B (sunlight) exposure. Vitamin D3, obtained from the isomerization of pre-vitamin D3 in the epidermal basal layers or intestinal absorption of natural and fortified foods and supplements, binds to vitamin D-binding protein (DBP) in the bloodstream, and is transported to the liver. D3 is hydroxylated by liver 25-hydroxylases (25-OHase). The resultant 25-hydroxycholecalciferol (25(OH)D3) is 1α-hydroxylated in the kidney by 25-hydroxyvitamin D3-1α-hydroxylase (1α-OHase). This yields the active secosteroid 1α,25(OH)2D3 (calcitriol), which has different effects on various target tissues23. The synthesis of 1α,25(OH)2D3 from 25(OH)D3 is stimulated by parathyroid hormone (PTH) and suppressed by Ca2+, Pi and 1α,25(OH)2D3 itself. The rate-limiting step in catabolism is the degradation of 25(OH)D3 and 1α,25(OH)2D3 to 24,25(OH)D3 and 1α,24,25(OH)2D3, respectively, which occurs through 24-hydroxylation by 25-hydroxyvitamin D 24-hydroxylase (24-OHase), encoded by the CYP24A1 gene. 24,25(OH)D3 and 1α,24,25(OH)2D3 are consequently excreted. The main effects of 1α,25(OH)2D3 on various target tissues are highlighted above.

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Bsml (A60890G)Tru9l (G61050A)Fokl (C27823T)Apal (G61888T) Taql (T61938C)

~75 kb

48 kDa

1f 1e 1a 1d 1c 2 3 4 5 6 7 8 91b

Chromosome 12VDR gene

VDR protein

q13–14

P PS51 S208

DNA binding(aa 24–90, 91–115)

Nuclear localization(aa 49–55, 79–105)Hormone ligand binding(aa 227–244, 268–316, 396–422)

Dimerization(aa 37, 91–92, 244–263, 317–395)Transactivation(aa 246, 416–422)

NAF-2

Hinge region1 24 49 91 115 227244268 317 396 422 427 aa

A/B E/FC D

activate the Raf–mitogen-activated protein kinase extracel-lular signal-regulated kinase kinase (MEK)–mitogen-acti-vated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) cascade in skeletal muscle cells74. Activation of the Raf–MEK–MAPK–ERK cascade, which mediates proliferative cellular effects, may be a response to increased Ca2+ in normal colon73 and skeletal muscle cells74, and may not have a direct role in the antiproliferative activities of 1α,25(OH)2D3 in tumour cells (discussed below). In addi-tion, ERK can also increase the transcriptional activity of the vDR75, and nongenomic activation of PKC may stabi-lize vDR (through phosphorylation)23,76, thereby affecting the transcriptional activity of the receptor. Therefore, the nongenomic activation of these pathways may cooperate with the classical genomic pathway to transactivate vDR and elicit the antiproliferative effects of 1α,25(OH)2D3, but this remains to be elucidated.

Anti-tumour effects of 1α,25(OH)2D3 signalling1α,25(OH)2D3 has been examined preclinically for its therapeutic efficacy in chemopreventive and anticancer activity. A chemoprevention study used Nkx3‑1;Pten mutant mice to recapitulate prostate carcinogenesis, and showed that 1α,25(OH)2D3 administration delayed the onset of prostate intraepithelial neoplasias (PIN) and had better anti-tumour activity when administered to mice with early-stage (PIN) rather than advanced-stage prostate disease77. Furthermore, studies using model systems of squamous cell carcinoma (SCC)78, prostate adenocarcinoma79, cancers of the ovary80, breast81 and lung82 showed that the administration of 1α,25(OH)2D3 or vitamin D analogues had significant anticancer effects. The effects of 1α,25(OH)2D3 and its derivatives have been shown to function through the vDR to regulate proliferation, apoptosis and angiogenesis62,83–87.

Box 1 | The vitamin D receptor

The human VDR gene (which encodes the vitamin D receptor), located on chromosome 12q, is composed of promoter and regulatory regions (1a–1f) and exons 2–9, which encode 6 domains (A – F) of the full length VDR protein (see figure)23. VDR nuclear localization signals (blue) direct the receptor into the nucleus155,156 along microtubule tracts to the nuclear pores157. Upon 1α,25(OH)2D3 binding to the hormone ligand-binding domain (red), VDR is stabilized by the phosphorylation of serine 51 in the DNA-binding domain (green) by protein kinase C76, and serine 208 in the hinge region by casein kinase II158. VDR associates with the retinoic acid receptor (RXR) through the dimerization domains (yellow). The 1α,25(OH)2D3–VDR–RXR complex binds to the vitamin D response elements (VDREs) through the DNA-binding domain in the promoters of target genes. Conformational change in the VDR results in the dissociation of the co-repressor, silencing mediator for retinoid and thyroid hormone receptors (SMRT), and allows interaction of the VDR activation function 2 (AF2) transactivation domain (light grey) with stimulatory coactivators, such as steroid receptor coactivators (SRCs), vitamin D receptor-interacting proteins complex and nuclear coactivator-62 kDa–Ski-interacting protein (NCoA62–SKIP)23 that mediate transcriptional activation.

Non-synonymous (FokI) and synonymous (BsmI, ApaI, TaqI and Tru9I) single-nucleotide polymorphisms (SNPs) have been identified in VDR (defined by restriction enzymes, polymorphisms are indicated in parentheses). FokI polymorphism at translation initiation codon results in a smaller VDR that interacts with transcription factor 2B (TF2B) more efficiently and has greater transcriptional activity than the full length VDR159. Although the functional effects of these SNPs remain unknown, they have been reported to be associated with increased susceptibility to primary and metastatic breast cancer17, squamous cell carcinoma160, colorectal cancer161,162, and prostate cancer163,164, but may be protective against head and neck cancer165.

The expression of VDR is an important determinant of the tumour cell response to 1α,25(OH)2D3. The VDR is overexpressed or repressed in several histological types of cancer (TABLE 3), demonstrating tissue-type variations in 1α,25(OH)2D3 signalling (supplemental information S1 (table)). VDR expression increases in hyperplastic polyps and in the early stages of tumorigenesis, but declines in late-stage poorly differentiated tumours and is absent in associated metastases. Tumours of the colon with the highest expression of VDR were most responsive to 1α,25(OH)2D3 treatment85,166. However, downregulation of the VDR in colon cancer cells through the transcription factor SNA1L167 reduces the anticancer effect of the vitamin D analogue EB1089.

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http://www.nature.com/nrc/journal/v7/n9/suppinfo/nrc2196.html


[cAMP]

CDKN1ACYP24A1 SPP1

ACPI3K

Raf isoforms

MEK1/2

Ras

ERK–MAPK1/2

PKA

PVDR

1α,25(OH)2D3

Ca2+

PLCγ

PKC

PVDR

memVDR

Caveolae

?

1α,25(OH)2D3

9cRARXR

9cRARXR P

VDR Nucleus

P

SRC-1

CBP/P300

PBAFSWI/SNF

9cRA

NCoA62–SKIP

5′ 3′

Chromatinremodeling(histoneacetylation)

VDREs

Transcriptionalactivation Cross-talk

P9cRA

NCoA62–SKIP

5′ 3′VDREs

RNAPol II

DRIPs

9cRARXR P

VDR

WSTF

WINACHDAC

complexes NCoR–SMRT

Chromatinremodelling(histonedeacetylation)

Transcriptionalrepression

CYP27B1 PTH

nVDREs

Generepression

Genetranscription

1α,25(OH)2D3

d

a

b

c TF2B

VDIR

1α,25(OH)2D3 GPCR

VDRRXR

VDRRXR

DRIP205

SOCchannels

Figure 2 | 1α,25(OH)2D3-mediated transcriptional regulation. Classical action of 1α,25(OH)2D3 is mediated by binding of the vitamin D receptor (VDR)−9-cis-retinoic acid receptor (RXR) complex at the vitamin-D response elements (VDREs). a | Transcriptional activation involves the co-activators, steroid receptor coactivators (SRCs), nuclear coactivator-62 kDa–Ski-interacting protein (NCoA62–SKIP) and histone acetyltransferases (HATs), CREB binding protein (CBP)–p300 and polybromo- and SWI-2-related gene 1 associated factor (PBAF–SNF) to acetylate histones to derepress chromatin. b | Binding of the vitamin D receptor-interacting protein 205 (DRIP205) to the activation function 2 (AF2) of VDR (and RXR) attracts a mediator complex containing other vitamin D receptor-interacting proteins (DRIPs) that bridge the VDR–RXR–NCoA62–SKIP–DRIP205 complex with transcription factor 2B (TF2B) and RNA polymerase II (RNA Pol II) for transcription initiation. The presence of the multiprotein complex facilitates increased transcription of genes, such as CDKN1A (which encodes the cyclin-dependent kinase inhibitor p21), CYP24A1 (which encodes 24-OHase) and SPP1 (which encodes osteopontin)23. c | 1α,25(OH)2D3-mediated transcriptional repression involves VDR–RXR heterodimer association with VDR-interacting repressor (VDIR) bound to E-box-type negative VDREs (nVDREs), dissociation of the HAT co-activator and recruitment of histone deacetylase (HDAC) co-repressor54. Williams syndrome transcription factor (WSTF) potentiates transrepression by interacting with a multifunctional, ATP-dependent chromatin-remodelling complex (WINAC) and chromatin55. This leads to the repression of genes, such as CYP27B1 (which encodes 1α-OHase) and PTH (which encodes parathyroid hormone). d | Non-genomic, rapid actions of 1α,25(OH)2D3 are hypothesized to involve 1α,25(OH)2D3 binding to cytosolic (VDR) and membrane VDR (memVDR), also found in caveolae, and speculated to activate the mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) 1 and 2 cascade68 through the phosphorylation (P) and activation of Raf by protein kinase C (PKC) by Ca2+ influx through store-operated Ca2+ (SOC) channels. 1α,25(OH)2D3 stimulates SOC Ca2+ influx (in muscle cells) by trafficking of the classic VDR to the plasma membrane, where the VDR interacts with the SOC channel. Ca2+ influx activates Ca2+ messenger systems, such as PKC. Activated PKC can phosphorylate VDR. 1α,25(OH)2D3 binding to G-protein coupled receptors (GPCRs) activates phospholipase Cγ (PLCγ), Ras, phosphatidylinositol 3-kinase (PI3K) and protein kinase A (PKA) pathways, and induces MAPK–ERK1 and 2 signalling. Activated Raf–MAPK–ERK may engage in cross-talk with the classical VDR pathway to modulate gene expression. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate.

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MYCTCF1CD44PARG

VDR

1α,25(OH)2D3

Nucleus

β-catenin

Frizzled

Wnt↑E-cadherinTGFβ

β-catenin

β-catenin

SMADs β-catenin

APC

Degradation

TCF4

p107/p130

E2F4/5

DP1 Cell cycle

Degradation

Growth arrest

TGFβ

R1TG

FβR2

EGFR

TGF1

R

IGF1

Ras

Raf isoforms

ERK–MAPK1/2

MEK1/2

P13K

Akt

BCL2

BCL-XL

BAX

BCL-XS

Effectorcaspases

Telomerase

Apoptosis

DifferentiationApoptosisGrowth inhibition

a b c d e f g

Cytosol

p15 p21 p27MYC

CDK4/6 CDK2

Cyclin D1,2,3 Cyclin E

pRB pRB

E2F1,2,3

+P P P

SKP2

Antiproliferative effects of 1α,25(OH)2D3. Cell-cycle perturbation is central to 1α,25(OH)2D3-mediated antiproliferative activity in tumour cells (supplemen-tal information S1 (table)). Progression through the cell cycle is regulated by cyclins, and their association with CDKs and CDK inhibitors (CKIs). Expression of the CKIs p21 and p27 inhibits proliferation, in part by inducing G1 cell-cycle arrest and withdrawal from the cell cycle (G0). CDKN1A and GADD45A contain a functional vDRE and are direct transcriptional tar-gets of 1α,25(OH)2D3–vDR. However, many genes are transcriptionally affected by 1α,25(OH)2D3 but do not

contain vDREs, and their transcriptional activation or repression may not be directly mediated by vDR. 1α,25(OH)2D3–vDR transcriptional activation of CDKN1A induces cell-cycle exit (differentiation) and cell-cycle arrest in human U937 myelomonocytic cells62. Treatment of human breast cancer MCF7 cells with 1α,25(OH)2D3 also increases the expression of CDKN1A and CDKN1B, (which encodes p27) and represses CCND1 (encoding cyclin D1), CCND3 (encoding cyc-lin D3), CCNA1 (which encodes cyclin A1) and CCNE1 (which encodes cyclin E1), and hence leads to the inhibi-tion of CDK activity and pRb hypophosphorylation88,89.

Figure 3 | Key cancer-related signalling pathways targeted by 1α,25(OH)2D3. 1α,25(OH)2D3 inhibits mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) 1 and 2 signalling through suppression of epidermal growth factor (EGFR; a) and insulin-like growth factor 1 (IGF1; b), which both target Ras. 1α,25(OH)2D3 induces apoptosis through the IGFR1−phosphatidylinositol 3-kinase (PI3K)−Akt-dependent signalling pathway (b), inhibiting telomerase (c), downregulating BCL2, inducing BAX and activating caspase cleavage (d). Cell-cycle progression is perturbed by 1α,25(OH)2D3 through S-phase kinase-associated protein ubiquitin ligase (SKP2; targeting p27 for degradation; e), and MYC, which results in pRB dephosphorylation; and transforming growth factor-β(TGFβ; f) cross-talk. Cell-cycle perturbation by 1α,25(OH)2D3 ultimately affects the association of retinoblastoma pocket proteins (pRB and p107/p130) and the E2F family of transcription factors and DP polypepitide (DP1) heterodimers that mediate the transcription of cell-cycle genes. Association of E2F1, 2 and 3 with pRB in its hypophosphorylated state and interaction of the E2F4 and 5 transcriptional repressors and DP1 with p107/p130 prevent transcription of cell-cycle genes and restrain cell-cycle progression. Activation of VDR by 1α,25(OH)2D3 induces the expression of E-cadherin (g), thereby promoting the translocation of β-catenin from the nucleus to the plasma membrane and competing with T-cell transcription factor 4 (TCF4) for β-catenin binding; thus inhibiting the Wnt–β-catenin–TCF4 signalling pathway, which leads to the induction of MYC, TCF1 (transcription factor 1), CD44 and PPARG (peroxisome proliferator-activated receptor-γ). APC, adenomatosis polyposis coli; CDK, cyclin-dependent kinase; pRB, phosphorylated retinoblastoma; Wnt, wingless-related MMTV integration site.

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Similarly, the treatment of SCC cells with 1α,25(OH)2D3 induces G0/G1 cell-cycle arrest owing to the tran-scriptional activation of CDKN1B and consequent pRb hypophosphorylation90. However, in this context CDKN1A expression was repressed, indicating that the cell-cycle arrest is an indirect effect of 1α,25(OH)2D3 treatment or that cell-type specificity might determine the ability of activated 1α,25(OH)2D3–vDR to induce CDKN1A expression90. Other genes have been shown to be transcriptionally affected by 1α,25(OH)2D3 in colon cancer, ovarian carcinoma and leukaemia cells, such as activation of GADD45 (REF 63), which is involved in DNA damage responses, repression of TYMS (which encodes thymidylate synthetase)91 and TK1 (which encodes thymidine kinase)91, which are involved in DNA replication, and activation of the INK4 fam-ily92 of cyclin D-dependent kinase inhibitors, which mediate G1 cell-cycle arrest; whereas cyclin E–CDK2 and the SKP2 (S-phase kinase-associated protein 2) ubiquitin ligase, which targets CKIs to the proteasome, are downregulated93 by 1α,25(OH)2D3. 1α,25(OH)2D3 treatment also results in the repression of the proto-oncogene MYC89,94, which significantly contributes to the antiproliferative effects of 1α,25(OH)2D3.

1α,25(OH)2D3 can have many indirect effects on cell-cycle regulation owing to cross-talk with other pathways; for example, 1α,25(OH)2D3 treatment can result in the upregulation of IGFBP3 (which encodes insulin growth factor binding protein 3) and trans-forming growth factor-β (TGFβ)–SMAD3 signalling cascades and by downregulating the epidermal growth factor receptor (EGFR) signalling pathway67,95,96 (FIG. 3). Although there appears to be an overall inhibition of cell-cycle progression in tumour cells treated with 1α,25(OH)2D3, the precise molecular basis for such an effect differs from one tumour cell type to another such that a unifying hypothesis with regard to the exact mechanism of 1α,25(OH)2D3-mediated cell-cycle perturbation has not been possible (supplemental information S1 (table)).

Activation of the vDR by 1α,25(OH)2D3 can also inhibit tumour cell proliferation by inducing differentia-tion in various myeloid leukaemia cell lines and freshly isolated leukaemia cells62,83, which is dependent on the formation of activated vDR and phosphatidylinositol 3-kinase (PI3K) complexes97. However, in haematopoeitic progenitor cells, 1α,25(OH)2D3 inhibits differentiation through vDR-independent suppression of interleukin 12 (IL12) protein secretion and down-regulation of other co-stimulatory molecules (CD40, CD80 and CD86)98. In cell lines of head and neck, colon and prostate tumours, administration of 1α,25(OH)2D3 or vitamin D analogues induces the expression of genes that are associated with the differentiated cell of origin91,99,100. In various colon cancer cells, treatment with 1α,25(OH)2D3 induces differentia-tion either by increasing PKC- and JNK-dependent JUN activation101 or by differentially regulating the expression of inhibitor of DNA binding 1 and 2 (ID1 and ID2), which encode proteins that are transcriptional regulators of epi-thelial cell proliferation (ID2) and differentiation (ID1); the repression of ID2 mediated the antiproliferative effects

of 1α,25(OH)2D3102. Recent findings reported by Palmer

et al.103 indicate that 1α,25(OH)2D3 promotes differentia-tion through the induction of CDH1 (which encodes E cadherin) in adenomatosis polyposis coli (APC)-mutated human colorectal cancer Sw480 cells. CDH1 activation consequently restrained cell growth by facilitating the translocation of β-catenin from the nucleus to the plasma membrane, thus inhibiting β-catenin-mediated tran-scription and allowing activated vDR to compete with β-catenin for transcription factor binding. Again, there appears to be no specific mechanism regarding the ability of 1α,25(OH)2D3 to induce differentiation in tumour cells (supplemental information S1 (table)).

Apoptosis. In addition to the antiproliferative effects of 1α,25(OH)2D3, there is increasing evidence that 1α,25(OH)2D3 exerts anti-tumour effects by regulat-ing key mediators of apoptosis, such as repressing the expression of the anti-apoptotic, pro-survival proteins BCL2 and BCL-XL, or inducing the expression of pro-apoptotic proteins (such as BAX, BAK and BAD). It has been reported that 1α,25(OH)2D3 downregulates BCL2 expression in MCF-7 breast tumour and HL-60 leukaemia cells and upregulates BAX and BAK expres-sion in prostate cancer, colorectal adenoma and carci-noma cells84. In addition to regulating the expression of the BCL2 family, 1α,25(OH)2D3 might also directly activate caspase effector molecules, although it is unclear whether 1α,25(OH)2D3-induced apoptosis is caspase-dependent84. In support of this idea, the treat-ment of mouse SCC tumour cells with 1α,25(OH)2D3 increased vDR expression and concomitantly inhib-ited the phosphorylation of ERK104. Upstream of ERK, the growth-promoting and pro-survival signalling molecule MEK is cleaved and inactivated in a caspase-dependent manner in cells that undergo apoptosis after treatment with 1α,25(OH)2D3. Recently, a novel mechanism of 1α,25(OH)2D3-mediated apoptosis in epithelial ovarian cancer cells was proposed by Jiang et al.105, wherein they showed that 1α,25(OH)2D3 destabilizes telomerase reverse transcriptase (TERT) mRNA, therefore inducing apoptosis through tel-omere attrition resulting from the down-regulation of telomerase activity. The diverse effects observed for 1α,25(OH)2D3-mediated apoptosis suggest that although anti-proliferative effects directed against the tumour are clear in vitro and in vivo (supple-mental information S1 (table)), dissecting the exact mechanism(s) central to these activities remains a challenge.

Angiogenesis. 1α,25(OH)2D3 inhibits the proliferation of endothelial cells in vitro and reduces angiogenesis in vivo106–108. vascular endothelial growth factor (vEGF)-induced endothelial cell tube formation and tumour growth are inhibited in vivo by 1α,25(OH)2D3 admin-istration to mice with vEGF-overexpressing MCF-7 xenografts86. 1α,25(OH)2D3 can increase VEGF mRNA levels in vascular smooth muscle cells109 and upregu-late mRNA levels of the potent anti-angiogenic factor thrombospondin 1 (THBS1) in Sw480-ADH human

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Platinum analoguesPlatinum‑based chemotherapeutics that crosslink DNA and therefore impair the progression of DNA replication machinery.

TaxanesDrugs that inhibit microtubule dynamics by stabilizing GDP‑bound tubulin. Microtubules form the mitotic spindle and so taxanes prevent the completional of mitosis.

MyelodysplasiaAny of a group of bone marrow disorders that have markedly abnormal reduction in one or more types of circulating blood cells owing to defective growth and maturation of blood‑forming cells in the bone marrow.

HypercalcemiaExcess of Ca2+ in the blood. Chronic elevated serum levels of Ca2+ (12.0 mg dL) can result in urinary calculi (renal or bladder stones) and abnormal heart rhythms. severe hypercalcemia (above 15–16 mg dL) can result in coma and cardiac arrest.

OsteodystrophyDefective bone ossification that occurs when the kidney fails to maintain proper levels of Pi and Ca2+. This results in slowed bone growth and causes bone deformities in children. In adults, renal osteodystrophy results in thin and weak bones, bone and joint pain and vulnerability to osteoporosis.

OsteoporosisA condition that is characterized by a decrease in bone mass with decreased density and enlargement of bone spaces producing porosity and brittleness of the bone.

PharmacokineticsThe characteristic interactions of a drug and the body in terms of its absorption, distribution, metabolism and excretion.

colon tumour cells102. In SCC cells, 1α,25(OH)2D3 induces the angiogenic factor interleukin 8 (IL8)110, but in prostate cancer cells 1α,25(OH)2D3 interrupts IL8 signalling leading to the inhibition of endothelial cell migration and tube formation111. A significant inhibition of metastasis is observed in prostate and lung murine models treated with 1α,25(OH)2D3, and these effects may be based, at least in part, on the anti-angiogenic effects described79,82. Interestingly, in tumour-derived endothelial cells (TDECs), 1α,25(OH)2D3 induces apop-tosis and cell-cycle arrest; however, these effects are not seen in endothelial cells isolated from normal tissues or from Matrigel plugs (Matrigel-derived endothelial cells)106. Recently, Chung et al.112 demonstrated that TDECs may be more sensitive to 1α,25(OH)2D3 owing to the epigenetic silencing of CYP24A1. Therefore, direct effects of 1α,25(OH)2D3 on endothelial cells may have a primary role in the 1α,25(OH)2D3-mediated anti-tumour activity that is observed in animal models of cancer.

Preclinical combination studiesIn vitro and in vivo analyses indicate that 1α,25(OH)2D3 acts synergistically with chemotherapeutic agents. 1α,25(OH)2D3 potentiates the anticancer activity of agents such as platinum analogues113–115, taxanes116,117 and DNA-intercalating agents117,118. Optimal potentiation is seen when 1α,25(OH)2D3 is administered before or simultaneously with chemotherapy treatment; admin-istration of 1α,25(OH)2D3 after the cytotoxic agent does not provide potentiation114,116. The combination of 1α,25(OH)2D3 and cisplatin in SCC cells in vitro induced tumour cell apoptosis characteristic of 1α,25(OH)2D3 alone. The pro-apoptotic signalling molecule MEKK1 (mitogen-activated protein kinase kinase kinase 1), is up-regulated in both apoptotic and pre-apoptotic SCC cells treated with 1α,25(OH)2D3 (REF 104). This up-regu-lation of MEKK1 was potentiated in combination with cisplatin treatment, suggesting that 1α,25(OH)2D3 pre-treatment commits cells to undergo apoptosis through specific molecular pathways (probably the MEK signal-ling pathway), and that this effect is increased when cells are treated with an additional genotoxic stimulus113. Similar effects are seen in MCF-7 cells treated with the vitamin D analogue ILX 23-7553 in combination with doxorubicin or ionizing radiation118. In these studies, ILX 23-7553 increased doxorubicin cytotoxicity and blocked the induction of p53 expression. Increased anti-tumour activity with 1α,25(OH)2D3 and the taxane paclitaxel is associated with a significant decrease in p21 expression, which sensitizes cells to both DNA-damaging agents (such as cisplatin and doxorubicin) and microtubule-disrupting agents (such as paclitaxel and docetaxel)116. In SCC and PC-3 (prostate cancer) xenografts, pre-treatment with 1α,25(OH)2D3 resulted in an increased anti-tumour effect in combination with paclitaxel116. Similar results have also been observed in vivo with MCF-7 xenografts in which mice were treated with vitamin D analogues and paclitaxel119. 1α,25(OH)2D3-mediated downregulation of cyclooxygenase 2 (COX2) expression in prostate cancer cells leads to decreased

prostaglandin activity, the induction of their degradation through the upregulation of 15-hydroxyprostaglandin dehydrogenase, and reduction of prostaglandin recep-tors120. These findings support the rationale for clinical evaluation of a combination of 1α,25(OH)2D3 and non-steroidal anti-inflammatory drugs (NSAIDs) for prostate cancer therapy120. Increased anti-tumour effects with 1α,25(OH)2D3 combination therapy offers the opportu-nity for the clinical use of 1α,25(OH)2D3 across several tumour types where modest effects are observed with chemotherapy alone.

Clinical trials of 1α,25(OH)2D3with the recognition of the preclinical antiproliferative and pro-differentiating effects of vitamin D in the 1970s and 1980s, a number of attempts were made to trans-late these findings into the clinic. Several investigators attempted to administer 1α,25(OH)2D3 as a differenti-ating agent in myelodysplasia and acute leukaemia121–123. Although some patients seemed to respond to the ther-apy, these improvements were not enough to encour-age further trials, as 20–30% of patients who received a daily dose of 1α,25(OH)2D3 developed hypercalcemia. Such findings have reinforced the conviction that less hypercalcemic analogues of vitamin D, with modified chemical structures to make them less prone to degra-dation by 24-OHase (FIG. 4a), must be developed if the therapeutic advantages of vitamin D biological effects are to be realized124,125. It is important to note that the early anticancer studies of 1α,25(OH)2D3 were conducted using dosing schedules optimized for the treatment of renal osteodystrophy and osteoporosis, and the doses important for anticancer effects were not investigated separately. Had the administration of 1α,25(OH)2D3 been developed from an anticancer standpoint, the following considerations would have been determined: first, optimal biologically-effective dose and maximum tolerated dose (MTD) across several cancers; second, the most effective dosing schedules to achieve antican-cer activity; third, 1α,25(OH)2D3-dependent signalling targets and molecular end-points; fourth, 1α,25(OH)2D3 interactions with other cytotoxic or other anticancer drugs that may be therapeutically advantageous; and finally, design of clinical trials that mirror, as much as possible, the exposures active in preclinical models to determine whether biological effects can be achieved in human tumours in clinical therapy (FIG. 4b).

Several studies have attempted to define a safe and effective clinical treatment regimen126–129. These investigations were based on the recognition that most positive preclinical studies used high-dose, intermit-tent 1α,25(OH)2D3. Although it is clear that 20–30% of patients receiving 1α,25(OH)2D3 at a dose of 1.5–2.0 µg a day develop hypercalcemia130, there have been few studies that have compared continuous and intermittent dosing regimens in cancer patients. Muindi and col-leagues131 have determined the pharmacokinetic profile of a 1α,25(OH)2D3 regimen that is active in a preclinical animal model. High-dose 1α,25(OH)2D3 (daily for 3 days 0.125 µg per mouse ~6.25 µg per kg (body weight)) resulted in growth inhibition of the syngeneic mouse SCC

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1α,25(OH)2D3 or new analoguesand drug combinations

Epidemiology/risk factors• Nutritional composition• UV exposure• VDR polymorphisms• Genetic background

Cancerpatient

Vitamin D levels(serum and cancer tissue)• Effectors of 1α,25(OH)2D3 metabolism: expression and activity of 25-OHase, 1α-OHase, 24-OHase, VDR

1α,25(OH)2D3 associated toxicities:Improve drug dosingPharmacokineticsPharmacodynamics

Clinical trials• Prevention• Anti-tumour therapy

Clinical assessment• Therapeutic impact,response and biomarkersevaluation

Mechanisms of action• Genomic (VDR)• Non-genomic (memVDR)• Apoptosis• Cell cycle• Angiogenesis• Cell signaling cross-talk• Cell–cell interaction

In vivo systems• Preclinical animal models:syngeneic, xenografts and genetically-modified (VDR–/–)

In vitro systems• Tumour cells• Stromal cells• Progenitor cancer stem cells

1 ,25(OH)2D3 or new analogues and drug combinations

Clinical dosingschedules

Prediction of 1α,25(OH)2D3 response

HO OH

OH

EB1089HO

OH

OHParicalcitol

HO OH

OH

ILX23-7553

O

HO

OH

OH OCT

HO

OH

25(OH)D3

25-HydroxycholecalciferolHO

OH

OH

1α,25(OH)2D3Calcitriol

LY2108491

SHO

OS

O

O

LY2109866

SHO

O

O

OH

LG190119OO

O O

Vitamin D analogues Vitamin D receptor modulators

a

b

HO

Vitamin D3

Cholecalciferol12

A

C D

Side chain

Seco-B-ring

3

4 10

5 19

67

8 15

16

17

18

11

12

13

9

2120 22

2324

25

14

Figure 4 | Development of 1α,25(OH)2D3 and vitamin D analogues as anticancer drugs. a | Cholecalciferol (vitamin D3) is 25-hydroxylated at C-25 (denoted by carbon atom number on the structure of cholecalciferol) to form 25-hydroxyc-holecalciferol (25(OH)D3). This is 1α-hydroxylated at C-1 by 1α-OHase to yield 1α,25(OH)2D3 (calcitriol). 1α,25(OH)2D3 is a secosteroid that is similar in structure to steroids but with a ‘broken’ B-ring (denoted seco-B-ring) where two of the carbon atoms (C-9 and C-10) of the four steroid rings are not joined. Many vitamin D analogues (left) retain the secosteroid structure with modified side chain structures around the C-24 position, which makes them less hypercalcemic and less prone to degradation by 24-OHase170,171. Several structures of vitamin D analogues referred to in the text are shown: paricalcitol (19-nor-1α(OH)2D2), ILX23-7553 (16-ene-23-yne-1α,25(OH)2D3), OCT (Maxacalcitol, 22-oxa-1α,25(OH)2D3) and EB1089 (Seocalcitol, 1α-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D3). Vitamin D receptor modulators (VDRMs, right) are non-secosteroidal in structure. Some of the representative compounds described are LY2108491, LY2109866 and LG190119 (REFs 146,147). b | Paradigm for development and clinical translation of 1α,25(OH)2D3 as an anticancer agent. Establishment of in vitro and in vivo experimental systems is crucial to developing 1α,25(OH)2D3 or vitamin D analogues that target vitamin D metabolism and signalling. These systems allow the mechanisms of action of 1α,25(OH)2D3 to be studied along with novel analogues (also in combination with cytotoxic drugs) in multiple transformed cell types and their biological effects (tumour and normal tissues) in animals. Importantly, studies on the pharmacokinetics and pharmacodynamics of drug action will enable the development of better designed clinical dosing schedules for clinical trials that will mirror the exposures active in preclinical models where optimal biological effects of 1α,25(OH)2D3 are demonstrated and are achievable in human tumours in clinical therapy.

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cells. At these doses, the area under the curve (AUC) 0–24 h (37 ± 2.5 ng•hr ml) and Cmax (22 nM) of 1α,25(OH)2D3 were within the concentration range that does not cause toxicity in patients131. Although SCC is a sensitive model, concentrations and exposure to 1α,25(OH)2D3 that inhibit SCC tumour growth are also active in many human tumour xenograft models41,78,80–82,132,133.

In developing an anticancer agent, more aggressive management of toxicity and use of supportive care approaches often allow one to overcome mild to mod-erate side effects. Beer and colleagues129 conducted a standard phase I dose-escalation trial of 1α,25(OH)2D3 administered orally once a week, and found that 2.8 µg per kg (body weight) can be safely administered with-out any side effects. Dose escalation was not continued because at doses higher than 2.4 µg per kg (body weight) oral absorption was found to be incomplete and unreli-able (BOX 2). Several phase I trials have been conducted in which an MTD of 1α,25(OH)2D3 has been sought127,134,135. Dose-limiting toxicity of oral 1α,25(OH)2D3 has not been observed in these studies.

As discussed above, combinations of 1α,25(OH)2D3 with other anticancer agents demonstrate synergistic interactions. Phase I studies of 1α,25(OH)2D3 plus paclitaxel127 and 1α,25(OH)2D3 plus gefitinib136 for the treatment of advanced malignancies, and phase II stud-ies of 1α,25(OH)2D3 plus carboplatin and 1α,25(OH)2D3 plus docetaxel for the treatment of prostate cancer137,138

have been completed (supplemental information S2 (table)). Most of these studies are based on persuasive preclinical data, but confront a problem not usually encountered in single-agent or combination phase I studies in cancer: dose, toxicity and pharmacokinetic data for 1α,25(OH)2D3 as a single agent are limited. Although investigators working with the Novacea formulation of 1α,25(OH)2D3 (DN-101) have defined suitable pharmacokinetics and have shown the feasibil-ity of very high doses of 1α,25(OH)2D3, an aggressive MTD in cancer patients has not been determined. Trump and colleagues139 completed a 43-patient study of 1α,25(OH)2D3 in escalating oral doses to a maximum of 12 µg 1α,25(OH)2D3 three times a week together with dexamethasone. Minimal hypercalcemia was observed and high-dose intermittent 1α,25(OH)2D3 plus dexamethasone was safe and feasible139. At present, dexamethasone in combination with weekly intra-venous 1α,25(OH)2D3 plus gefitinib is being explored in a phase I clinical trial to determine whether an aggres-sive MTD can be achieved (supplemental information S2 (table)). Glucocorticoids are used clinically to amelio-rate hypercalcemia in a number of situations including 1α,25(OH)2D3 intoxication140. Dexamethasone also sig-nificantly improves 1α,25(OH)2D3 anti-tumour efficacy, in vitro and in vivo, through direct effects on the vDR141. In studies of tumour-bearing animals, dexamethasone increases vDR receptor number without changing the ligand affinity (Kd) in SCC tumour tissue xenograft and the kidney, but not in gastrointestinal mucosa141. The ability of dexamethasone to increase anti-tumour activity in certain tissues and decrease toxicity is medi-ated through the modulation of vDR expression. The combined use of a glucocorticoid and 1α,25(OH)2D3 is a viable approach to reducing side-effects experienced by patients treated with 1α,25(OH)2D3.

Another approach to reducing potential toxicity and increasing anti-tumour activity is the development of vitamin D analogues and vitamin D receptor modu-lators (vDRMs) (FIG. 4a) that are less prone to cause hypercalcemia. However, considerable data indicate that when 1α,25(OH)2D3 is given at an intermittent schedule, clinical use is not limited by hypercalcemia or hypercalciuria126–129,137,138,142. vitamin D analogues have been synthesized and their properties examined143. Although many appear to be less hypercalcemogenic than 1α,25(OH)2D3, the complexities of in vitro–in vivo data and dose extrapolation limit the conclusions that analogues which cause less hypercalcemia are equipo-tent in terms of anticancer effects. For example, most analogues that cause less hypercalcemia bind less tightly to the vDR, a property that probably reduces their anti-tumour effects143. As non-steroidal tissue-selective oestrogen-receptor (ER) modulators (SERMs) such as tamoxifen have proven clinically successful for the prevention of breast cancer in high-risk women, and for the treatment of ER-positive breast cancer144, recent development of non-secosteroidal vDRMs have shown potential in anticancer therapy145. The novel non-secos-teroidal vDRMs LY2108491 and LY2109866 (FIG. 4a) were identified as potent tissue-selective agonists in

Area under the curve(AUC). In pharmacokinetics, the area under the curve is a plot of concentration of drug in serum over time that represents the measure of an individual’s exposure to the drug.

BioavailabilityMeasurement of an administered dose of a therapeutically active drug that reaches the systemic circulation and depends on the mode of administration.

Cmax

Maximum or ‘peak’ concentration of a drug observed after its administration.

GlucocorticoidsCorticosteroids are involved in carbohydrate, protein and fat metabolism to regulate liver glycogen and blood sugar by increasing gluconeogenesis; clinically used for anti‑inflammatory and immunosuppressive effects.

Box 2 | The pharmacology of 1α,25(OH)2D3

In developing an agent for human use, it is crucial to understand the pharmacokinetics and, when possible, the pharmacodynamic parameters associated with drug administration. Although the pharmacodynamics of 1α,25(OH)2D3 have not been well studied, the pharmacokinetics of 1α,25(OH)2D3 have been extensively investigated using a standard, commercial formulation of 1α,25(OH)2D3 (Rocaltrol, Hoffman–LaRoche). Importantly, these pharmacokinetic studies revealed that dose escalation did not result in the escalation of systemic exposure. Both groups found that the desirable linear relationship between dose administered and systemic exposure (area under the curve (AUC) and Cmax) was lost at doses >16 µg127,129,131. In addition, there was marked variation (5–10×) in AUC and Cmax among patients receiving the same dose of 1α,25(OH)2D3. Two approaches indicate that these pharmacokinetic findings are a product of the pharmaceutical characteristics of Rocaltrol when administered at high dose rather than reduced absorption or increased catabolism of 1α,25(OH)2D3 within the patients studied. Novocea, Inc.168 has developed a new formulation of 1α,25(OH)2D3 (DN-101, Ascentar) for high-dose applications (15 µg and 45 µg caplets) and pharmacokinetic studies indicate a linear relationship between dose and exposure at oral doses up to 168 µg, indicating that there is no ‘barrier’ to gastrointestinal absorption of 1α,25(OH)2D3 at the doses studied168.

High-dose intravenous 1α,25(OH)2D3 (Calcijex, Abbott Pharmaceuticals) has been investigated in a phase I clinical trial136 (supplemental information S2 (table)). A linear relationship between dose and exposure was observed across a wide dose range (10–125 µg), indicating that 1α,25(OH)2D3 administration is not associated with rapid induction of 1α,25(OH)2D3 catabolism. The same patients monitored on multiple occasions had no convincing evidence that the pharmacokinetics of 1α,25(OH)2D3 on day 1 of a once a day for 3 days a week schedule are different from the pharmacokinetics on day 28; neither does the administration of either paclitaxel or dexamethasone modify 1α,25(OH)2D3 pharmacokinetics. Although formal bioavailability studies of 1α,25(OH)2D3 have not been done, inspection of pharmacokinetic curves in our studies of intravenous 1α,25(OH)2D3 and those of the DN-101 study suggest that oral absorption of a suitable formulation is very efficient (80–90%) even at a high dose136,169.

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keratinocytes, human peripheral blood mononuclear cells and osteoblasts, but have weak potency in intestinal cells146. Furthermore, the non-secosteroidal, tissue-selec-tive vDRMs were less calcemic in vivo compared with 1α,25(OH)2D3, and show efficacy in an animal model of psoriasis146; however, their potential in anticancer therapy has not been determined. Polek and collegues reported that a novel vDRM, LG190119 (FIG. 4a), inhibited LNCaP xenograft tumour growth without hypercalcemia147. Non-secosteroidal vDRMs represent promising therapeutic agents for the treatment of cancers with tissue-selectivity and potential evasion of hypercalcemia.

Clinical studies of vitamin D analogues have focused primarily on continuous daily administration of EB1089 (FIG. 4a; seocalcitol, 1α-dihydroxy-22,24-diene-24,26,27-trihomovitamin D3) to patients with breast cancer, colorectal cancer or hepatocellular carcinoma148–150. EB1089 failed to show evidence of anti-tumour activity in these studies, and potentially problematic hypercalcemia was seen but was not dose limiting. Paricalcitol (FIG. 4a; 19-nor-1α,25-(OH)2D2, Zemplar), an analogue developed by Abbott, appears to be more effective than 1α,25(OH)2D3 in the man-agement of renal osteodystrophy and chronic renal disease151, and preclinical data indicate that paricalcitol has anti-tumour effects in prostate, pancreas, lung and breast cancers, as well as multiple myeloma152. Current phase I clinical trials have been initiated for paricalcitol plus gemcitabine and paricalcitol plus zoledronic acid (a bisphosphonate) in patients with advanced solid tumours and multiple myeloma, respectively, to estab-lish whether very high doses of these analogues can be safely administered intravenously when an intermit-tent schedule is used (supplemental information S2 (table)).

In patients with prostate cancer that progresses despite castration (so-called androgen-independent prostate cancer or AIPC), 1α,25(OH)2D3 has been stud-ied in ‘standard’ dose and schedule and at a high dose. The most striking indication of 1α,25(OH)2D3 anti-tumour effects in AIPC is the randomized trial reported by Beer et al.138 that used docetaxel (36 µg once a week) and 1α,25(OH)2D3 (DN-101, 45 µg one day before docetaxel). The survival in the DN-101 plus docetaxel-treated patients was improved, but further confirmation is required because survival was not the primary end point of this phase II study. Novacea is currently con-ducting a 1,000 patient phase III trial to further evaluate this survival difference. It is also striking that severe or life-threatening side effects, including thromboembolic complications, were reduced in the DN-101 arm153. The results of this trial point to potentially clinically relevant anti-tumour effects of 1α,25(OH)2D3 in combination with docetaxel.

Conclusions and future perspectivesThe data described above support the continued exploration of vitamin D supplementation and 1α,25(OH)2D3 as approaches to cancer prevention and treatment, respectively. The epidemiological data indicate that vitamin D deficiency is relatively com-

mon, at least in some parts of the US and Europe, and that inadequate levels of 25(OH)D3 are associated with an increased risk and poor prognosis of several types of cancer16. In view of the numerous other potential consequences of vitamin D deficiency to human health, such as rickets and osteomalacia, one could easily rec-ommend more aggressive monitoring of 25(OH)D3 levels as part of a health maintenance programme. Meta-analysis and cancer-prevention trials indicate that vitamin D3 supplementation to achieve a level of >82 nmol per L 25(OH)D3 can lower the incidence of colorectal cancer by 50%20. To achieve serum levels in this range, individuals require a daily 4,000 IU (inter-national unit) supplement of vitamin D3 (REF. 20), which is achievable with the current formulations that range from 200–2,000 IU and a liquid formulation of 2,000 IU per drop. Formal randomized studies to optimize replacement strategies and to evaluate vitamin D3 as a cancer-preventative approach should be considered.

Changes in the expression of proteins important in vitamin D synthesis and catabolism (25-OHase, 1α-OHase, 24-OHase) and those crucial for mediat-ing the biological effects of 1α,25(OH)2D3 (vDR) have been shown to be associated with poor differentiation status and prognosis of several types of cancer, such as colon cancer31–33,35. The overexpression of vitamin D catabolic enzymes in cancer suggests that low cellular 1α,25(OH)2D3 is also associated with poor prognosis, but this has not yet been addressed convincingly. In addition, the steady-state level of cellular 1α,25(OH)2D3 in tumour tissue is difficult to measure. Assessment of the catabolic enzyme that degrades 1α,25(OH)2D3, such as 24-OHase (encoded by CYP24A1), may have merit in the development of prognostic models. Indeed, CYP24A1 is overexpressed in many cancers (TABLE 3). Of interest, epigenetic silencing of CYP24A1 in tumour-derived endothelial cells renders the tumour sensitive to the anti-angiogenic effects of 1α,25(OH)2D3 (REF. 112). various molecules can inhibit 24-OHase (such as azoles and vitamin D analogues). These merit exploration and further development as specific small-molecule 24-OHase inhibitors, especially in combi-nation with high-dose intermittent 1α,25(OH)2D3 or other vitamin D analogues. These may maximize intracellular 1α,25(OH)2D3 content and exert optimal antiproliferative effects.

The growth restraining, differentiation and apop-tosis-inducing effects of 1α,25(OH)2D3 in different tumour cell types is well documented (supplemental information S1 (table)). Across various tumour cell lines, different molecular markers of cell cycle, differ-entiation and apoptosis can be observed with no clear pattern of modulation by 1α,25(OH)2D3; perhaps these studies demonstrate the importance of heterogeneity to the 1α,25(OH)2D3 response, even among similar tumour cell types. The antiproliferative actions of 1α,25(OH)2D3 may depend on the differentiation status of the tumour cells and vDR expression level, as well as genomic or post-translational modifications of co-activator proteins that are essential for the assembly of the transcriptionally active vDR complex154.

1α,25(OH)2D3 intoxicationThe symptoms of hypervitaminosis D (excessive doses of vitamin D) are a result of hypercalcemia caused by increased intestinal Ca2+ absorption. Gastrointestinal symptoms include anorexia, nausea and vomiting.

HypercalciuriaExcessive urinary Ca2+ excretion. The morbidity associated with hypercalciuria is related to kidney stone disease and bone demineralization leading to osteopaenia (decrease in bone density) and osteoporosis.

Thromboembolic complicationsAssociated with blockage of a blood vessel by a particle that has dislodged from a blood clot at its primary formation site.

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An important study that shows the importance of 1α,25(OH)2D3 and its regulated synthesis in colon mucosa; expression of 1α-OHase is lost in undifferentiated, malignant human colon cancer.

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1α,25(OH)2D3 has prominent antiproliferative, anti-angiogenic and pro-differentiative effects in a broad range of cancers (supplemental information S1 (table)). These effects are mediated through perturba-tion of several important signalling pathways medi-ated through genomic and non-genomic mechanisms. 1α,25(OH)2D3 potentiates the anti-tumour effects of many anticancer therapeutic compounds, and sev-eral clinical trials indicate that the administration of high-dose 1α,25(OH)2D3 and vitamin D analogues is safe and feasible. Clear delineation of the MTD of

1α,25(OH)2D3 is required so that more extensive tri-als of 1α,25(OH)2D3, alone and in combination with cytotoxic and other anticancer agents, can be con-ducted. Initial phase II studies and a single phase III study suggest that 1α,25(OH)2D3 is an active agent in cancer therapy for prostate cancer (supplemental information S2 (table)). The ease of administration, broad biological effects of 1α,25(OH)2D3 and the potentiation of several anticancer drugs strongly sup-port the continued development of 1α,25(OH)2D3 as an anticancer drug.

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83. Wang, X. & Studzinski, G. P. Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1α, 25-dihydroxyvitamin D3-induced differentiation of HL60 cells. J. Cell Biochem. 80, 471–482 (2001).

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AcknowledgementsD.L.T. and C.S.J. are supported by grants from the US National Cancer Institute, Department of Defense, American Cancer Society and The Roswell Park Alliance Foundation. C.S.J. is also supported by the Robert, Lew and Ann Wallace Endowment Fund.

Competing interests statementThe authors declare competing financial interests, see web version for details.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBCL2 | BGLAP | CBP | CCNA1 | CCNC | CCND1 | CCND3 | CCNE1 | CDH1 | CDKN1A | CDKN1B | COX2 | CYP24A1 | CYP27A1 | CYP27B1 | EGFR | GADD45 | HDAC | ID1 | ID2 | IGFBP3 | MEKK1 | MYC | PI3K | PTH | SKIP | SKP2 | SMAD3 | SMRT | TGFβ | THBS1 | TF2B | TK1 | TYMS | VDR | VEGF | WSTFNational Cancer Institute: http://www.cancer.govbreast cancer | colorectal cancer | prostate cancer

FURTHER INFORMATIONAuthors’ homepage: http://roswellpark.org

SUPPLEMENTARY INFORMATIONSee online article: S1 (table) | S2 (table)

all linKs are acTiVe in THe Online PDf

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Vitamin D signalling pathways in cancer: potential for anticancer